Nucleic Acids Encoding Anthelmintic Agents And Plants Made Therefrom

ABSTRACT

The present invention provides DNA constructs, transgenic plants containing such constructs, and methods of making the plants. The DNA constructs encode a polypeptide that when expressed results in the production of fatty acid compounds having anthelmintic activity. Transgenic plants expressing such a polypeptide can exhibit enhanced resistance to plant parasitic nematodes, particularly when expressed in vegetative tissues. Transgenic plants expressing such a polypeptide can also be useful for non-pesticidal industrial uses.

This application is a divisional of U.S. application Ser. No.10/912,534, filed Aug. 4, 2004, which is a continuation-in-part (andclaims the benefit of priority under 35 USC 120) of U.S. applicationSer. No. 10/772,227, filed Feb. 4, 2004, all of which are incorporatedby reference in their entireties.

FIELD OF THE INVENTION

This invention relates to the field of plant pathology and plant genetictransformation. More particularly, the invention relates to methods andcompositions for the increased production of novel fatty acids intransgenic plants for industrial purposes including controlling plantpathogens such as plant-parasitic nematodes.

BACKGROUND OF THE INVENTION

Nematodes (derived from the Greek word for thread) are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved to be very successful parasitesof both plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control. CAB International, NewYork).

Nematode parasites of plants can inhabit all parts of plants, includingroots, developing flower buds, leaves, and stems. Plant parasites areclassified on the basis of their feeding habits into the broadcategories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

The application of chemical nematicides remains the major means ofnematode control. However, in general, chemical nematicides are highlytoxic compounds known to cause substantial environmental impact and areincreasingly restricted in the amounts and locations in which they canbe used. For example, the soil fumigant methyl bromide which has beenused effectively to reduce nematode infestations in a variety ofspecialty crops, is regulated under the U.N. Montreal Protocol as anozone-depleting substance and is scheduled for elimination in 2005 inthe US (Carter (2001) California Agriculture, 55(3):2). It is expectedthat strawberry and other commodity crop industries will besignificantly impacted if a suitable replacement for methyl bromide isnot found. Similarly, broad-spectrum nematicides such as Telone (variousformulations of 1,3-dichloropropene) have significant restrictions ontheir use because of toxicological concerns (Carter (2001) CaliforniaAgriculture, Vol. 55(3): 12-18).

The macrocyclic lactones (e.g., avermectins and milbemycins), as well asdelta-endotoxins from Bacillus thuringiensis (Bt), are chemicals that inprinciple provide excellent specificity and efficacy which should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two nematicidal agents have provenless effective in agricultural applications against root pathogens.Although certain avermectins show exquisite activity against plantparasitic nematodes these chemicals are hampered by poor bioavailabilitydue to their light sensitivity, tight binding to soil particles anddegradation by soil microorganisms (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).Consequently despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

Bt delta endotoxins must be ingested to affect their target organ, thebrush border of midgut epithelial cells (Marroquin et al. (2000)Genetics. 155(4): 1693-1699). Consequently they are not anticipated tobe effective against the dispersal, non-feeding, juvenile stages ofplant parasitic nematodes in the field. Because juvenile stages onlycommence feeding when a susceptible host has been infected, nematicidesmay need to penetrate the plant cuticle to be effective. Transcuticularuptake of a 65-130 kDa protein—the size of typical Bt delta endstoxins—is unlikely. Furthermore, soil mobility is expected to berelatively poor. Even transgenic approaches are hampered by the size ofBt delta toxins because delivery in planta is likely to be constrainedby the exclusion of large particles by the feeding tubes of certainplant parasitic nematodes such as Heterodera (Atkinson et al. (1998)Engineering resistance to plant-parasitic nematodes. In: The Physiologyand Biochemistry of Free-Living and Plant-parasitic Nematodes (eds R. N.Perry & D. J. Wright), CAB International 1998).

Fatty acids are another class of natural compounds that have beeninvestigated as alternatives to the toxic, non-specific organophosphate,carbamate and fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this predicted mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including herbicides (e.g., SCYTHE by Dow Agrosciences isthe C9 saturated fatty acid pelargonic acid), bactericides, fungicides(U.S. Pat. Nos. 4,771,571; 5,246,716), and insecticides (e.g., SAFERINSECTICIDAL SOAP by Safer, Inc.).

The phytotoxicity of fatty acids has been a major constraint on theirgeneral use in post-plant agricultural applications (U.S. Pat. No.5,093,124) and the mitigation of these undesirable effects whilepreserving pesticidal activity is a major area of research. Post-plantapplications are desirable because of the relatively short half-life offatty acids under field conditions.

The esterification of fatty acids can significantly decrease theirphytotoxicity (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359). Suchmodifications can however lead to loss of nematicidal activity as isseen for linoleic, linolenic and oleic acid (Stadler et al. (1994)Planta Medica 60(2):128-132) and it may be impossible to completelydecouple the phytotoxicity and nematicidal activity of pesticidal fattyacids because of their non-specific mode of action. Perhaps notsurprisingly, the nematicidal fatty acid pelargonic acid methyl ester(U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) shows a relativelysmall “therapeutic window” between the onset of pesticidal activity andthe observation of significant phytotoxicity (Davis et al. (1997) JNematol 29(4S):677-684). This is the expected result if both thephytotoxicity and the nematicidal activity derive from the non-specificdisruption of plasma membrane integrity.

Ricinoleic acid, the major component of castor oil, has been shown tohave an inhibitory effect on water and electrolyte absorption usingeverted hamster jejunal and ileal segments (Gaginella et al. (1975) JPharmacol Exp Ther 195(2):355-61) and to be cytotoxic to isolatedintestinal epithelial cells (Gaginella et al. (1977) J Pharmacol ExpTher 201(1):259-66). These features are likely the source of thelaxative properties of castor oil which is given as a purgative inhumans and livestock (e.g., castor oil is a component of some de-wormingprotocols because of its laxative properties). In contrast, the methylester of ricinoleic acid is ineffective at suppressing water absorptionin the hamster model (Gaginella et al. (1975) J Pharmacol Exp Ther195(2):355-61).

It has been reported that short- and medium-chain fatty acids and salts(e.g., C6 to C12) have superior fungicidal activity (U.S. Pat. Nos.5,093,124 and 5,246,716). Not surprisingly, the commercial fungicidaland moss killing product De-Moss comprises mainly fatty acids and saltsin this size range. The phytotoxicity of these shorter fatty acids alsomakes them suitable as broad-spectrum herbicides when used at higherconcentrations as is exemplified by the commercial herbicide SCYTHEwhich comprises the C9 fatty acid pelargonic (nonanoic) acid. U.S. Pat.Nos. 5,093,124, 5,192,546, 5,246,716 and 5,346,698 teach that C16 to C20fatty acids and salts such as oleic acid (C18:1) are suitableinsecticidal fatty acids. Insecticidal fatty acid products such asM-PEDE and SAFER Insecticidal Concentrate whose active ingredientscomprise longer chain fatty acids rich in C16 and C18 componentsrepresent real world applications of this scientific information. Incontrast, the prior art provides little guidance for the selection ofsuitable broad-spectrum nematicidal fatty acids and what informationexists is often contradictory.

Stadler and colleagues (Stadler et al. (1994) Planta Medica 60(2):128-132) tested a series of fatty acids against L4 and adult C. elegansand found that a number of common longer chain fatty acids such aslinoleic (C18:2), myristic (C14:0), palmitoleic (C16:1) and oleic(C18:1) acids had significant nematicidal activity. C. elegans was notvery sensitive to C6 to C10 (medium chain) fatty acids. Stadler et al.commented that their results contrasted with those of an earlier studyon the plant parasite Aphelenchoides besseyi where C8 to C12 fatty acidswere found to be highly active while linoleic acid—a C18 fattyacid—showed no activity. The differential sensitivity of specificnematodes to various fatty acids is again evident in the study of Djianand co-workers (Djian et al. (1994) Pestic. Biochem. Physiol.50(3):229-239) who demonstrate that the nematicidal potency of shortvolatile fatty acids such as pentanoic acid can vary between species(e.g., Meloidogyne incognita is over a hundred times more sensitive thanPanagrellus redivivus). The recent finding by Momin and Nair (Momin &Nair (2002) J. Agric. Food Chem. 50(16):4475-4478) that oleic acid at100 μg/mL over 24 hours is not nematicidal to either Panagrellusredivivus or Caenorhabditis elegans further confuses the situation as itdirectly conflicts with the LD50 of 25 μg/mL (LD90 100 μg/mL) measuredby Stadler and coworkers.

In summary, unlike the case for fungicides, herbicides and insecticides,the prior art provides no specific or credible guidance to aid in theselection of suitable nematicidal fatty acids. Moreover, whereasDe-Moss, SCYTHE, M-PEDE and SAFER, are examples of successful pesticidalfatty acid products in these three areas respectively, there arecurrently no examples of commercial nematicidal fatty acid products inwidespread use.

Many plant species are reported to be highly resistant to nematodes. Thebest documented of these include marigolds (Tagetes spp.), rattlebox(Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castorbean (Ricinus communis), margosa (Azardiracta indica), and many membersof the family Asteraceae (family Compositae) (Hackney & Dickerson.(1975) J Nematol 7(1):84-90). In the case of the Asteraceae, thephotodynamic compound alpha-terthienyl has been shown to account for thestrong nematicidal activity of the roots. Castor beans are plowed underas a green manure before a seed crop is set. However, a significantdrawback of the castor plant is that the seed contains toxic compounds(such as ricin) that can kill humans, pets, and livestock and is alsohighly allergenic. In many cases however, the active principle(s) forplant nematicidal activity has not been discovered and it thereforeremains difficult to derive commercially successful nematicidal productsfrom these resistant plants or to transfer the resistance toagronomically important crops such as soybeans and cotton.

Genetic resistance to certain nematodes is available in some commercialcultivars (e.g., soybeans), but these are restricted in number and theavailability of cultivars with both desirable agronomic features andresistance is limited. The production of nematode resistant commercialvarieties by conventional plant breeding based on genetic recombinationthrough sexual crosses is a slow process and is often further hamperedby a lack of appropriate germplasm.

Small chemical effectors can have significant advantages where sizeexclusion of larger molecules is a concern (e.g., with sedentary plantparasitic nematodes). However, unless the small molecule nematicidalactive has high in planta mobility, or the chemical stimulates increasedsystemic resistance, a transgene encoding an enzyme must still beexpressed in an appropriate spatial and temporal manner to be effective.With many plant parasitic nematodes this means that root expression ofthe nematicidal product is likely important for nematode control. It hasbeen reported that when a constitutive promoter such as a CauliflowerMosaic Virus (CaMV) 35S promoter is used to drive expression of certainhydroxylase enzymes, no significant amounts of protein production orhydroxylase activity is observed in non-seed tissues (e.g., roots orleaves), nor do hydroxylated fatty acids accumulate (van de Loo et al.(1995) Proc Natl Acad Sci USA 92(15):6743-7; Broun & Sommerville (1997)Plant Physiol. 113(3):933-942; Broun et al. (1998) Plant J.13(2):201-210; U.S. Pat. No. 6,291,742; U.S. Pat. No. 6,310,194).

There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

SUMMARY OF THE INVENTION

The invention concerns DNA constructs that include sequences encodingfatty acid hydroxylases or epoxygenases, transgenic plants harboringsuch constructs, and methods for making such transgenic plants. Thesetransgenic plants can exhibit increased resistance to nematodes and canbe useful for controlling nematodes in an environmentally safe manner.The invention is based in part on the surprising discovery that certainhydroxylated or epoxygenated fatty acids and methyl esters (e.g.,ricinoleate, vernolate), exhibit nematicidal activity. These fatty acidsshow significantly enhanced nematicidal activity over other eighteencarbon free fatty acids such as oleate, elaidate and linoleate. Nucleicacids encoding hydroxylase or epoxygenase polypeptides can be introducedinto plants in order to increase the levels of hydroxylated orepoxygenated fatty acids and thus aid in controlling nematode damage incommercially important plant species. These novel hydroxylase andepoxygenase constructs are also useful for increasing the accumulationof hydroxy and epoxy fatty acids for other industrial uses (e.g.,providing safe sources of ricinoleic acid).

In one aspect, the invention features a transgenic plant containing atleast one DNA construct. The construct comprises at least one regulatoryelement that confers expression in vegetative tissues of a plant. Theregulatory element is operably linked to a nucleic acid encoding apolypeptide that is effective for catalysing the conversion of asubstrate to a C16, C18, or C20 monounsaturated fatty acid product. TheC16-C20 monounsaturated fatty acid product can be:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na+, phosphatidylcholine, or phosphatidylethanolamine,wherein both R₁ and R₂ are hydroxyl, one of R₁ and R₂ is hydroxyl andthe other is hydrogen, or one of R₁ and R₂ is keto and the other ishydrogen, and wherein R₃ is C2, C4, or C6 alkyl. The C16-C20monounsaturated fatty acid product can also be:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na+, phosphatidylcholine, or phosphatidylethanolamine, andwherein R₃ is C2, C4, or C6 alkyl.

The C═C double bond can be cis or trans. The R₃ moiety of the C16-C20monounsaturated fatty acid product can be C2 alkyl. A C16-C20monounsaturated fatty acid product can have hydroxy, hydrogen, and C4alkyl as the R₁, R₂ and R₃ moieties, respectively, e.g., a ricinoleateproduct. Alternatively, a C16-C20 monounsaturated fatty acid product canhave an epoxy moiety at the 12^(th) and 13^(th) carbons counting fromthe carbonyl carbon and C4 alkyl at R₃, e.g., a vernolate product.

The plant can have an increased amount of a hydroxy-fatty acid, e.g.,ricinoleic acid, in a vegetative tissue, relative to a correspondingplant that lacks the DNA construct. The hydroxy-fatty acid canconstitute from about 0.01% to about 25% of the total fatty acid contentof the tissue. In some embodiments, the plant has an increased amount ofan epoxy-fatty acid, e.g., vernolic acid, in a vegetative tissue,relative to a corresponding plant that lacks the DNA construct. Theepoxy-fatty acid can constitute from about 0.01% to about 25% of thetotal fatty acid content of the tissue.

The regulatory element can be a 5′-regulatory element or a 3′-regulatoryelement. The regulatory element can confer expression in root tissue, orin leaf tissue. For example, a 5′-regulatory element can be a CaMV 35Spromoter, a potato ribosomal protein S27a Ubi3 promoter, an alfalfahistone H3.2 promoter, an IRT2 promoter, an RB7 promoter, an ArabidopsisFAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, a Ubi3 5′-UTR, an alfalfahistone H3.2 5′-UTR, or a CaMV35S 5′-UTR.

There can be more than one regulatory element operably linked to thepolypeptide coding sequence in the DNA construct. For example, a DNAconstruct can have two 5′-regulatory elements. The first 5′-regulatoryelement can be a Ubi3 promoter and the second 5′-regulatory element canbe an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, a potatoribosomal protein S27a Ubi3 5′-UTR, or a CaMV35S 5′-UTR. In someembodiments the DNA construct has a 5′-regulatory element and a3′-regulatory element. The 3′-regulatory element can be a Ubi3terminator or an E9 pea terminator. Alternatively, the 5′-regulatoryelement can be an Arabidopsis FAD2 5′-UTR or an Arabidopsis FAD3 5′-UTRand the 3′-regulatory element can be an Arabidopsis FAD2 3′-UTR or anArabidopsis FAD3 3′-UTR.

The DNA construct in a plant can include a nucleic acid that encodes aPDAT or DAGAT or lipase polypeptide, operably linked to one or moreregulatory elements that confer expression in vegetative tissues of aplant. Alternatively, the PDAT or DAGAT or lipase coding sequence andregulatory element can be part of a separate DNA construct in the plant.In some embodiments, the plant contains a DNA construct encoding adelta-12 or delta-15 fatty acid desaturase.

The amino acid sequence of the polypeptide can be SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, C.palaestina epoxygenase GenBank® No. CAA76156, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, a C.palaestina epoxygenase chimera, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ IDNO: 41, SEQ ID NO: 42, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136,SEQ ID NO: 137 or SEQ ID NO: 138. The nucleic acid encoding thepolypeptide can be SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 129, SEQ ID NO: 130,SEQ ID NO: 131, SEQ ID NO: 132 or SEQ ID NO: 133.

The plant can be a monocotyledonous or a dicotyledonous plant. Forexample, the plant can be a soybean, corn, cotton, rice, tobacco,tomato, wheat, banana, carrot, potato, strawberry or turf grass plant.

In another aspect, the invention features a method of making atransgenic plant. The method comprises obtaining a DNA construct asdescribed herein, and introducing the construct into a plant. The DNAconstruct can include nucleic acids encoding the polypeptides describedherein, and can include the regulatory elements described herein.

The invention also features a method of screening a transgenic plant foranthelmintic activity. The method comprises contacting a transgenicplant with a nematode under conditions effective to determine whether ornot the plant has anthelmintic activity. For example, the nematodes canbe contacted with one or more roots of the transgenic plant. Thetransgenic plant has a DNA construct that includes nucleic acidsencoding a hydroxylase or epoxygenase polypeptide described herein, andcan include the regulatory elements described herein. The method canalso be carried out with plant tissue, e.g., root tissue, leaf tissue orstem tissue from such a transgenic plant.

In another aspect, the invention features an isolated nucleic acid. Thenucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 25, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 129, SEQ ID NO: 130,SEQ ID NO: 131, SEQ ID NO: 132 or SEQ ID NO: 133.

In another aspect, the invention features a recombinant nucleic acidconstruct. The construct comprises at least one regulatory element thatconfers expression in vegetative tissues of a plant. The regulatoryelement is operably linked to a nucleic acid having the nucleotidesequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 129, SEQ ID NO:130, SEQ ID NO: 131, SEQ ID NO: 132 or SEQ ID NO: 133. The regulatoryelement can confer expression in, for example, roots or leaves. Theregulatory element can be a 5′-regulatory element having the nucleotidesequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44. The nucleic acidconstruct can further comprise a 3′-regulatory element having thenucleotide sequence set forth in SEQ ID NO: 45.

The invention also features a transgenic plant harboring a DNAconstruct. The construct comprises a nucleic acid encoding a fatty acidepoxygenase polypeptide or a fatty acid hydroxylase polypeptide,operably linked to a regulatory element conferring expression of thepolypeptide in a vegetative tissue of the plant. The polypeptide canhave the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, C.palaestina epoxygenase (GenBank® No. CAA76156), SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 134, SEQ ID NO: 135,SEQ ID NO: 136, SEQ ID NO: 137 or SEQ ID NO: 138.

The plant can have a significantly increased amount of a hydroxy-fattyacid, e.g., ricinoleic acid, in a vegetative tissue of the plantrelative to a corresponding plant that lacks the DNA construct. Thehydroxy-fatty acid can constitute from about 0.1% to about 10% of thetotal fatty acid content of the tissue. In some embodiments, the planthas a significantly increased amount of an epoxy-fatty acid, e.g.,vernolic acid, in a vegetative tissue of the plant relative to acorresponding plant that lacks the DNA construct. The epoxy-fatty acidcan constitute from about 0.1% to about 10% of the total fatty acidcontent of the tissue.

In another aspect, the invention features a transgenic plant containingat least one DNA construct. The construct comprises at least oneregulatory element that confers expression in at least one tissue ofseeds of a plant. The regulatory element is operably linked to a nucleicacid encoding a polypeptide that is effective for catalysing theconversion of a substrate to a C16, C18, or C20 monounsaturated fattyacid product. The C16-C20 monounsaturated fatty acid product can be:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na+, phosphatidylcholine, or phosphatidylethanolamine,wherein both R₁ and R₂ are hydroxyl, one of R₁ and R₂ is hydroxyl andthe other is hydrogen, or one of R₁ and R₂ is keto and the other ishydrogen, and wherein R₃ is C2, C4, or C6 alkyl. The C16-C20monounsaturated fatty acid product can also be:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na+, phosphatidylcholine, or phosphatidylethanolamine, andwherein R₃ is C2, C4, or C6 alkyl.

The C═C double bond can be cis or trans. The R₃ moiety of the C16-C20monounsaturated fatty acid product can be C2 alkyl. A C16-C20monounsaturated fatty acid product can have hydroxy, hydrogen, and C4alkyl as the R₁, R₂ and R₃ moieties, respectively, e.g., a ricinoleateproduct. Alternatively, a C16-C20 monounsaturated fatty acid product canhave an epoxy moiety at the 12^(th) and 13^(th) carbons counting fromthe carbonyl carbon and C4 alkyl at R₃, e.g., a vernolate product.

The regulatory element can be a 5′-regulatory element. The plant canhave an increased amount of a hydroxy-fatty acid, e.g., ricinoleic acid,in at least one tissue of seeds, relative to a corresponding plant thatlacks the DNA construct. In some embodiments, the plant has an increasedamount of an epoxy-fatty acid, e.g., vernolic acid, in at least onetissue of seeds, relative to a corresponding plant that lacks the DNAconstruct.

A “purified polypeptide”, as used herein, refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it is naturally associated. The polypeptide can constitute atleast 10, 20, 50, 70, 80 or 95% by dry weight of the purifiedpreparation.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicnucleic acid fragment, a fragment produced by polymerase chain reaction(PCR), or a restriction fragment; and (d) an engineered nucleic acidsuch as a recombinant DNA molecule that is part of a hybrid or fusionnucleic acid, (e.g., a gene encoding a fusion protein). Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones in a DNA library such as a cDNA or genomic DNAlibrary, or other nucleic acid existing among hundreds to millions ofother nucleic acids within, for example, gel slices containing a genomicDNA restriction digest. Although the phrase “nucleic acid molecule”primarily refers to the physical nucleic acid molecule and the phrase“nucleic acid sequence” refers to the sequence of the nucleotides in thenucleic acid molecule, the two phrases can be used interchangeably.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The term “ectopic expression” refers to a pattern of subcellular,cell-type, tissue-type and/or developmental or temporal expression thatis not normal for the particular gene or enzyme in question. It alsorefers to expression of a heterologous gene; e.g. a gene not naturallyoccurring in the organism (also termed “transgene” as described below).Such ectopic expression does not necessarily exclude expression innormal tissues or developmental stages.

As used herein, the term “transgene” means a nucleic acid that is partlyor entirely heterologous, i.e., foreign, to the transgenic plant,animal, or cell into which it is introduced, or, is homologous to anendogenous gene of the transgenic plant, animal, or cell into which itis introduced, but which is inserted into the plant's genome in such away as to alter the genome of the cell into which it is inserted (e.g.,it is inserted at a location which differs from that of the natural geneor its insertion results in a knockout). A transgene can include one ormore regulatory elements operably linked to a polypeptide codingsequence.

As used herein, the term “transgenic cell” refers to a cell containing atransgene. As used herein, a “transgenic plant” is any plant in whichone or more, or all, of the cells of the plant include a transgene. Atransgene may be integrated within a chromosome, or it may beextrachromosomally replicating DNA.

The terms “operably linked”, “operably inserted” or “operablyassociated” mean that a regulatory element is positioned in a DNAconstruct relative to a polypeptide coding sequence so as to effectexpression of the polypeptide.

As used herein, the terms “hybridizes under stringent conditions” and“hybridizes under high stringency conditions” refers to conditions forhybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60° C. or65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6×SSC buffer atabout 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at50° C.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

As used herein, the term “binding” refers to the ability of a firstcompound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, and 0.1 nM or less.

As used herein, the term “binds specifically” refers to the ability ofan antibody to discriminate between a target ligand and a non-targetligand such that the antibody binds to the target ligand and not to thenon-target ligand when simultaneously exposed to both the given ligandand non-target ligand, and when the target ligand and the non-targetligand are both present in molar excess over the antibody.

As used herein, the term “altering an activity” refers to a change inlevel, either an increase or a decrease in the activity, (e.g., anincrease or decrease in the ability of the polypeptide to bind orregulate other polypeptides or molecules) particularly a fatty aciddesaturase-like or fatty acid desaturase activity (e.g., the ability tointroduce a double bond at the delta-12 position of a fatty acid). Thechange can be detected in a qualitative or quantitative observation. Ifa quantitative observation is made, and if a comprehensive analysis isperformed over a plurality of observations, one skilled in the art canapply routine statistical analysis to identify modulations where a levelis changed and where the statistical parameter, the p value, is, forexample, less than 0.05.

Unless otherwise specified, a “substituted” carbon, carbon chain, ormethyl, alkyl can have one or more hydrogens replaced by another group,e.g., a halogen or a hydroxyl group.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, examples and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of drawings depicting the structures of ricinoleic acid,ricinelaidic acid, 12-oxo-9(Z)-octadecenoic acid,12-oxo-9(E)-octadecenoic acid, (12,13)-epoxy-trans-9-octadecenoic acidand vernolic acid. The numbering of the carbons is indicated with thecarbonyl (carboxyl) carbon being carbon 1. R═OH (acid); OCH₃ (methylester); O⁻Na⁺ (sodium salt).

FIG. 2 is an alignment of the sequences of the hydroxylase andepoxygenase polypeptides (SEQ ID NOs.: 13 to 24; 34 to 42) and A.thaliana (SEQ ID NO: 125), B. napus (SEQ ID NO: 126), G. max (SEQ ID NO:127) and S. indicum (SEQ ID NO: 128) FAD2 delta-12 desaturasepolypeptides (gi|15229956|ref|NP_(—)187819.1, gi|8705229|gb|AAF78778.1,gi|904154|gb|AAB00860.1 and gi|8886726|gb|AAF80560.1 respectively).

FIG. 3 is a schematic representation of transgenic epoxygenase andhydroxylase constructs. HA refers to the amino acid sequence YPYDVPDYA(SEQ ID NO: 139), which corresponds to residues 99-107 of humaninfluenza virus hemagglutinin. LB and RB refer to the left and rightborders, respectively, of an Agrobacterium T-DNA.

FIG. 4 is a schematic representation of the plasmid pUCAP6.

FIG. 5 is a schematic representation of the plasmid pUCAP4.

FIG. 6 is a schematic representation of the plasmid pUCAP3.

DETAILED DESCRIPTION

The present invention describes genes and genetic constructs encodingpolypeptides effective for producing small molecule chemicals that showsurprising nematicidal activity. The nematicidal activity is due in partto selective inhibition of metabolic processes that appear to beessential to nematodes and are either absent or non-essential invertebrates and plants. The invention therefore provides urgently neededDNA constructs, transgenic plants and methods of making such plants forenvironmentally safe control of plant-parasitic nematodes.

Fatty Acids

Unsaturated fatty acids are essential to the proper functioning ofbiological membranes. At physiological temperatures, polar glycerolipidsthat contain only saturated fatty acids cannot form theliquid-crystalline bilayer that is the fundamental structure ofbiological membranes. The introduction of an appropriate number ofdouble bonds (a process referred to as desaturation) into the fattyacids of membrane glycerolipids decreases the temperature of thetransition from the gel to the liquid-crystalline phase and providesmembranes with necessary fluidity. Fluidity of the membrane is importantfor maintaining the barrier properties of the lipid bilayer and for theactivation and function of certain membrane bound enzymes. There is alsoevidence that unsaturation confers some protection to ethanol andoxidative stress, suggesting that the degree of unsaturation of membranefatty acids has importance beyond temperature adaptation. Unsaturatedfatty acids are also precursors of polyunsaturated acids (PUFAs)arachidonic and eicosapentaenoic acids in animals, which are importantsources of prostaglandins. These molecules are local hormones that alterthe activities of the cells in which they are synthesized and inadjoining cells, mediating processes in reproduction, immunity,neurophysiology, thermobiology, and ion and fluid transport.

The ability of cells to modulate the degree of unsaturation in theirmembranes is primarily determined by the action of fatty aciddesaturases. Desaturase enzymes introduce unsaturated bonds at specificpositions in their fatty acyl chain substrates, using molecular oxygenand reducing equivalents from NADH (or NADPH) to catalyze the insertionof double bonds. In many systems, the reaction uses a short electrontransport chain consisting of NAD(P)H, cytochrome b5 reductase, andcytochrome b5, to shuttle electrons from NAD(P)H and the carbon-carbonsingle bond to oxygen, forming water and a double bond (C═C). Manyeukaryotic desaturases are endoplasmic reticulum (ER) bound non-hemediiron-oxo proteins that contain three conserved histidine-rich motifsand two long stretches of hydrophobic residues. These hydrophobic alphahelical domains are thought to position the protein with its bulkexposed to the cytosolic face of the ER and to organize the active sitehistidines to appropriately coordinate the active diiron-oxo moiety.

While most eukaryotic organisms, including mammals, can introduce adouble bond into an 18-carbon fatty acid at the Δ9 position, mammals areincapable of inserting double bonds at the Δ12 or Δ15 positions. Forthis reason, linoleate (18:2 Δ9,12) and linolenate (18:3 Δ9,12,15) mustbe obtained from the diet and, thus, are termed essential fatty acids.These dietary fatty acids come predominately from plant sources, sinceflowering plants readily desaturate the Δ12 and the Δ15 positions.Certain invertebrate animals, including some insects and nematodes, cansynthesize de novo all of their component fatty acids, includinglinoleate and linolenate. The nematode C. elegans, for example, cansynthesize de novo a broad range of polyunsaturated fatty acidsincluding arachidonic acid and eicosapentaenoic acids, a feature notshared by either mammals or flowering plants (Spychalla et al. (1997)Proc. Natl. Acad. Sci. USA 94(4):1142-7).

The C. elegans desaturase gene fat2 has been expressed in S. cerevisiaeand shown to be a delta-12 fatty acid desaturase (Peyou-Ndi et al.(2000) Arch. Biochem. Biophys. 376(2):399-408). This enzyme introduces adouble bond between the 12th and the 13th carbons (from the carboxylateend) and can convert the mono-unsaturated oleate (18:1 Δ9) andpalmitoleate (16:1 Δ9) to the di-unsaturated linoleate (18:2 Δ9,12) and16:2 Δ9,12 fatty acids, respectively.

The nematode delta-12 enzymes are potentially good targets foranti-nematode compounds for several reasons. Firstly, as mentionedabove, mammals are thought not to have delta-12 fatty acid desaturases.In addition, the nematode enzymes appear to be phylogenetically distinctfrom their homologs in plants, having less than 40% pairwise sequenceidentity at the amino acid level and phylogenetic analyses demonstrateclustering of nematode delta-12 and ω-3 desaturases away from homologsin plants. Experiments with both transgenic Arabidopsis and soybeansreveal that plants can tolerate significant reductions in linoleate orlinolenate, suggesting that inhibitors of delta-12 desaturases wouldlikely not be toxic to plants (Miquel & Browse (1992) J. Biol. Chem.267(3):1502-9; Singh et al. (2000) Biochem. Society Trans. 28: 940-942;Lee et al. (1998) Science 280:915-918). Thus, inhibitors of the enzymeare likely to be non-toxic to mammals.

We made the surprising discovery that the parent fatty acids and methylesters of certain fatty acid analogs (e.g., ricinoleate, vernolate) arenematicidal and have activity consistent with that of specificinhibitors of nematode delta-12 desaturases. The fatty acids and methylesters show significantly increased anthelmintic activity compared toeighteen carbon free fatty acids and esters such as oleate, elaidate andlinoleate. In contrast to short chain fatty acids and esters such aspelargonate (pelargonic acid or methyl pelargonate), fatty acid analogsthat are predicted delta-12 desaturase inhibitors show reducedphytotoxicity and can therefore be used effectively while minimizingundesirable damage to non-target organisms. Suitable nematode-inhibitorycompounds include compounds having the following fatty acids in free oresterified form: ricinoleic acid (12-hydroxoctadec-cis-9-enoic acid),hydroxypalmitoleic acid (12-hydroxyhexadec-cis-9-enoic acid),ricinelaidic acid, vernolic acid ((12,13)-epoxy-octadec-cis-9-enoicacid), and 12-oxo-9(Z)-octadecenoic acid.

Polypeptides

A polypeptide suitable for use in the invention is effective forcatalysing the conversion of a substrate to a C16, C18, or C20monounsaturated fatty acid product, e.g., a hydroxylated fatty acid oran epoxygenated fatty acid. The enzymatic products of hydroxylase orepoxygenase enzymes useful in the invention typically are fatty acids16, 18, or 20 carbons in length, or analogs thereof. Such productstypically have a cis (Z) or a trans (E) carbon double bond at thedelta-9 position, between C9 and C10 counting from the carbonyl(carboxyl) carbon. Such products also have hydroxy or epoxymodifications at C12, C13 or both C12 and C13. A fatty acid hydroxylaseor epoxygenase of this invention includes a polypeptide thatdemonstrates the ability to catalyze the production of ricinoleic,lesquerolic, hydroxyerucic (16-hydroxydocos-cis-13-enoic acid) orhydroxypalmitoleic (12-hydroxyhexadec-cis-9-enoic) from Coenzyme A, acylcarrier protein (ACP) or lipid-linked monoenoic fatty acid substratesunder suitable conditions.

In some embodiments, the product is a C16-C20 monounsaturated oxo-fattyacid that has the following structure:

One or both of R₁ and R₂ can be hydroxyl, e.g., R₁ is hydrogen and R₂ ishydroxyl, R₁ is hydroxyl and R₂ is hydrogen, or both R₁ and R₂ arehydroxyl. Alternatively, R₁ can be keto and R₂ hydrogen, or R₁ can behydrogen and R₂ keto. R₃ can be C2 alkyl, C4 alkyl, or C6 alkyl.

In other embodiments, the product is a C16-C20 epoxy monounsaturatedfatty acid product that has the following structure:

If X is hydrogen in the structures given above, the product is a freefatty acid. However, X can also be CoA, ACP, phosphatidylcholine, orphosphatidylethanolamine. X can also be glycerol, a glyceride, methyl,or Na⁺. In both of the structures given above, the double bond betweenthe 9^(th) and 10^(th) carbons can be cis or can be trans.

Whether a polypeptide exhibits hydroxylase activity or epoxygenaseactivity can be determined by testing the polypeptide e.g., in ahydroxylase assay described in U.S. Pat. No. 6,310,194, or anepoxygenase assay described in U.S. Pat. No. 6,329,518. A rapid andefficient method to identify suitable polypeptides is an analysis offatty acid production in yeast that express the polypeptide to betested. Since Saccharomyces cerevisiae does not produce linoleic acid(the substrate of delta-12 desaturase-like epoxygenases), linoleic acidor methyl linoleate is provided exogenously as a substrate. Anyconversion of the substrate to a hydroxylated or epoxygenated productcan be measured by, for example, gas chromatography-mass spectrometry(GC-MS) of total fatty acids after hydrolysis and conversion to methylesters. A polypeptide is considered to have hydroxylase activity orepoxygenase activity when it produces an amount of hydroxy- orepoxy-fatty acid that is statistically significantly greater inSaccharomyces cerevisiae that express the polypeptide, relative to theamount produced in corresponding control S. cerevisiae that lack or donot express the polypeptide. An alternative technique for identifyingsuitable polypeptides is an analysis of fatty acid content in vegetativetissues or at least one tissue of seeds of Arabidopsis plants, e.g.,leaf tissue, root tissue, or endosperm or embryo tissue.

Typically, a difference is considered statistically significant a p<0.05with an appropriate parametric or non-parametric statistic, e.g.,Chi-square test, Student's t-test, Mann-Whitney test, or F-test. In someembodiments, a difference is statistically significant at p<0.01,p<0.005, or p<0.001. A statistically significant difference in, forexample, the level of ricinoleic acid in seeds from a transgenicArabidopsis plant that expresses a hydroxylase polypeptide, compared tothe level in a control Arabidopsis plant, indicates that expression ofthe polypeptide results in an increase in the level of ricinoleic acid.The significantly increased amount of a hydroxy-fatty acid canconstitute from about 0.01% to about 25% by weight of the total fattyacid content of a sample, e.g., from about 0.03% to about 20%, about0.05% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%,about 0.2% to about 3%, about 0.5% to about 5.0%, about 0.5% to about10%, about 2.0% to about 15%, about 1.0% to about 5.0%, about 1.0% toabout 10%, about 3% to about 8%, about 3% to about 10%, about 4% toabout 9%, about 4% to about 13%, about 5% to about 20%, about 5% toabout 15%, or about 5% to about 10%. The significantly increased amountof an epoxy-fatty acid can constitute from about 0.01% to about 35% byweight of the total fatty acid content of a sample, e.g., from about0.03% to about 25%, about 0.05% to about 20%, about 0.1% to about 5%,about 0.2% to about 3%, about 0.5% to about 5.0%, about 0.5% to about10%, about 2.0% to about 15%, about 1.0% to about 5.0%, about 1.0% toabout 10%, about 3% to about 8%, about 3% to about 10%, about 4% toabout 9%, about 4% to about 13%, about 5% to about 20%, about 5% toabout 15%, or about 5% to about 10%.

In some embodiments, the polypeptide is a hydroxylase encoded by a geneisolated from Lesquerella or Ricinus plants. In other embodiments, thepolypeptide is an epoxygenase encoded by a gene isolated from Stokesia,Crepis or Vernonia plants. Examples of these enzymes include the oleatehydroxylases from Ricinus communis, Lesquerella fendleri, Lesquerellalindheimeri, Lesquerella gracilis and linoleate epoxygenases fromStokesia laevis, Crepis biennis, Crepis palaestina and Vernoniagalamensis.

In some embodiments, a polypeptide suitable for use in the invention isa fusion of two or more naturally-occurring amino acid sequences. Forexample, a naturally occurring oleate hydroxylase polypeptide derivedfrom Ricinus communis, Lesquerella fendleri, Lesquerella lindheimeri, orLesquerella gracilis can have approximately thirty amino acids at theN-terminus replaced by N-terminal amino acids from the Arabidopsisthaliana FAD2 gene. See, e.g., SEQ ID NOs: 19 through 23. Alternatively,a fusion polypeptide can be a naturally occurring linoleate epoxygenasederived from Stokesia laevis or Crepis biennis (e.g., SEQ ID NO: 24)where amino acids at the N-terminus are replaced by N-terminal aminoacids from the Arabidopsis thaliana FAD2 gene.

Other naturally occurring hydroxylases and epoxygenases are obtainableusing the specific exemplified sequences provided herein. Furthermore,it will be apparent that one can make synthetic hydroxylases havingmodified amino acid sequences. Modified amino acid sequences includesequences which have been mutated, truncated, increased and the like,whether such sequences were partially or wholly synthesized.

In some embodiments, a hydroxylase or epoxygenase suitable for use inthe invention has at least 60% overall amino acid sequence identity witha target polypeptide, e.g., 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99%sequence identity.

A percent sequence identity for any subject nucleic acid or amino acidsequence (e.g., any of the hydroxylase polypeptides described herein)relative to another “target” nucleic acid or amino acid sequence can bedetermined as follows. Such identity is calculated by determining thenumber of matched positions in aligned nucleic acid sequences, dividingthe number of matched positions by the total number of alignednucleotides, and multiplying by 100. A matched position refers to aposition in which identical nucleotides occur at the same position inaligned nucleic acid sequences. Percent sequence identity also can bedetermined for any amino acid sequence. To determine percent sequenceidentity, a target nucleic acid or amino acid sequence is compared tothe identified nucleic acid or amino acid sequence using the BLAST 2Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTN version 2.0.14 and BLASTP version 2.0.14. Thisstand-alone version of BLASTZ can be obtained from Fish & Richardson'sweb site (World Wide Web at “fr” dot “com” slash “blast”) or the U.S.government's National Center for Biotechnology Information web site(World Wide Web at “ncbi” dot “nlm” dot “nih” dot “gov”). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to -1; -r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt-j c:\seq2.txt -p blastn -o c:\output.txt -q -1-r 2. If the targetsequence shares homology with any portion of the identified sequence,then the designated output file will present those regions of homologyas aligned sequences. If the target sequence does not share homologywith any portion of the identified sequence, then the designated outputfile will not present aligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides from the target sequence presented in alignmentwith sequence from the identified sequence starting with any matchedposition and ending with any other matched position. A matched positionis any position where an identical nucleotide is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides. Likewise, gaps presentedin the identified sequence are not counted since target sequencenucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (i) a 500 amino acid target sequence is compared to asubject amino acid sequence, (ii) the Bl2seq program presents 200 aminoacids from the target sequence aligned with a region of the subjectsequence where the first and last amino acids of that 200 amino acidregion are matches, and (iii) the number of matches over those 200aligned amino acids is 180, then the 500 amino acid target sequencecontains a length of 200 and a sequence identity over that length of 90%(i.e., 180, 200×100=90). In some embodiments, the amino acid sequence ofa polypeptide suitable for use in the invention has 40% sequenceidentity to the amino acid sequence of SEQ ID NOS: 13, 14, 15, 16, 17,18, 36, 134, 135, 136, 137, or 138. In other embodiments, the amino acidsequence of a polypeptide suitable for use in the invention has greaterthan 40% sequence identity (e.g., >40%, >50%, >60%, >70%, >80%, >90%,or >95%) to the amino acid sequence of SEQ ID NOS: 13, 14, 15, 16, 17,18, 36, 134, 135, 136, 137, or 138.

It will be appreciated that different regions within a single nucleicacid target sequence that aligns with an identified sequence can eachhave their own percent identity. It is noted that the percent identityvalue is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13,and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18,and 78.19 are rounded up to 78.2. It also is noted that the length valuewill always be an integer.

The identification of conserved regions in a template, or subject,polypeptide can facilitate homologous polypeptide sequence analysis.Conserved regions can be identified by locating a region within theprimary amino acid sequence of a template polypeptide that is a repeatedsequence, forms some secondary structure (e.g., helices and betasheets), establishes positively or negatively charged domains, orrepresents a protein motif or domain. See, e.g., the Pfam web sitedescribing consensus sequences for a variety of protein motifs anddomains at http://www.sanger.ac.uk/Pfam/ andhttp://genome.wustl.edu/Pfam/. A description of the information includedat the Pfam database is described in Sonnhammer et al. (1998) Nucl.Acids Res. 26: 320-322; Sonnhammer et al. (1997) Proteins 28:405-420;and Bateman et al. (1999) Nucl. Acids Res. 27:260-262. From the Pfamdatabase, consensus sequences of protein motifs and domains can bealigned with the template polypeptide sequence to determine conservedregion(s).

Conserved regions also can be determined by aligning sequences of thesame or related polypeptides from closely related plant species. Closelyrelated plant species preferably are from the same family.Alternatively, alignments are performed using sequences from plantspecies that are all monocots or are all dicots. In some embodiments,alignment of sequences from two different plant species is adequate. Forexample, sequences from canola and Arabidopsis can be used to identifyone or more conserved regions.

Typically, polypeptides that exhibit at least about 35% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related proteins sometimes exhibit at least 40% amino acidsequence identity (e.g., at least 50%, at least 60%; or at least 70%, atleast 80%, or at least 90% amino acid sequence identity). In someembodiments, a conserved region of target and template polypeptidesexhibit at least 92, 94, 96, 98, or 99% amino acid sequence identity.Amino acid sequence identity can be deduced from amino acid ornucleotide sequence.

A polypeptide useful in the invention optionally can possess additionalamino acid residues at the amino-terminus or the carboxy-terminus. Forexample, 6×His-tag or FLAG™ residues can be linked to a polypeptide atthe amino-terminus. See, e.g., U.S. Pat. Nos. 4,851,341 and 5,001,912.As another example, a reporter polypeptide such as green fluorescentprotein (GFP) can be fused to the carboxy-terminus of the polypeptide.See, for example, U.S. Pat. No. 5,491,084.

Nucleic Acids

Among the nucleic acids suitable for the invention are those that encodea polypeptide described herein. Typically, such a nucleic acid isincorporated into a DNA construct suitable for introduction into a plantand integration into a plant genome. A DNA construct comprising anucleic acid encoding a hydroxylase or epoxygenase polypeptide isoperably linked to one or more regulatory elements that conferexpression in vegetative tissues or at least one tissue of seeds of aplant. Typically, a DNA construct includes a 5′-regulatory element and a3′-regulatory element for expression in transformed plants. In someembodiments, such constructs are chimeric, i.e., the coding sequence andone or more of the regulatory sequences are from different sources. Forexample, a polypeptide coding sequence can be a Ricinus communishydroxylase and a 5′-regulatory element can be a potato S27a promoter.However, non-chimeric DNA constructs also can be used. DNA constructscan also include cloning vector nucleic acids. Cloning vectors suitablefor use in the present invention are commercially available and are usedroutinely by those of ordinary skill in the art.

Regulatory elements typically do not themselves code for a gene product.Instead, regulatory elements affect expression of the coding sequence,i.e., transcription of the coding sequence, and processing andtranslation of the resulting mRNA. Examples of regulatory elementssuitable for use in a DNA construct include promoter sequences, enhancersequences, response elements or inducible elements that modulateexpression of a nucleic acid sequence. As used herein, “operably linked”refers to positioning of a regulatory element in a construct relative toa nucleic acid coding sequence in such a way as to permit or facilitateexpression of the encoded polypeptide. The choice of element(s) that areincluded in a construct depends upon several factors, including, but notlimited to, replication efficiency, selectability, inducibility, desiredexpression level, and cell or tissue specificity.

Suitable regulatory elements include promoters that initiatetranscription only, or predominantly, in certain cell types. Forexample, promoters specific to vegetative tissues such as groundmeristem, vascular bundle, cambium, phloem, cortex, shoot apicalmeristem, lateral shoot meristem, root apical meristem, lateral rootmeristem, leaf primordium, leaf mesophyll, or leaf epidermis can besuitable regulatory elements. A cell type or tissue-specific promotercan drive expression of operably linked sequences in tissues other thanvegetative tissue. Thus, as used herein a cell type or tissue-specificpromoter is one that drives expression preferentially in the targettissue, but can also lead to some expression in other cell types ortissues as well. Methods for identifying and characterizing promoterregions in plant genomic DNA include, for example, those described inthe following references: Jordano et al. (1989) Plant Cell, 1:855-866;Bustos et al. (1989) Plant Cell, 1:839-854; Green et al. (1988) EMBO J.7:4035-4044; Meier et al. (1991) Plant Cell, 3:309-316; and Zhang et al.(1996) Plant Physio. 110: 1069-1079.

Other suitable regulatory elements can be found in 5′-untranslatedregions (5′-UTR) and 3′-untranslated regions (3′-UTR). The terms 5′-UTRand 3′-UTR refer to nucleic acids that are positioned 5′ and 3′ to acoding sequence, respectively, in a DNA construct and that can be foundin mRNA 5′ to the initiation codon and 3′ to the stop codon,respectively. A 5′-UTR and a 3′-UTR can include elements that affecttranscription of the coding sequence, as well as elements that affectprocessing of mRNA and translation of the coding sequence.

Regulatory elements suitable for use in plants include nopaline andmannopine synthase regulatory elements, cauliflower mosaic virus 35Spromoters, Arabidopsis root periphery IRT2 promoter, Solanum tuberosum(potato) ribosomal S27a Ubi3 promoter, rice Actin I gene promoter andUbiquitin I gene promoter from maize (McElroy et al. (1995) Mol. Breed.1:27-37). Inducible nematode responsive promoters of interest includethe tobacco tobRB7 (Yamamoto et al. (1991) Plant Cell, 3(4):371-382),sunflower Sun-RB7 (Sarda et al. (1999) Plant Mol Biol. 40(1):179-191)and potato potRB7 (Heinrich et al. (1996) Plant Physiol. 112(2):861-864)promoters. Other exemplary promoter-5′-UTR constructs which can be usedin applications requiring root expression are listed in Table 8.

For embodiments where expression of a polypeptide is desired invegetative plant tissues such as leaves or roots, the use of all or partof the 5′ upstream non-coding regions (5′-UTR) and 3′ downstreamnon-coding regions (3′-UTR) of a Arabidopsis FAD2 or FAD3 gene arecontemplated. Also suitable is the construction of chimeric hydroxylasesand epoxygenases by swapping approximately the first 30 amino acids froma desaturase such as the FAD2 or FAD3 desaturases for the equivalentN-terminal region of the hydroxylase or epoxygenase as in the nucleicacids of SEQ ID NOs: 7 to 12 and the amino acid sequences of SEQ IDNOs.: 19 to 24. Particularly desirable are the use of chimericdesaturase-like epoxygenases or hydroxylases with non-seed specificUTRs.

Regulatory elements such as transcript termination regions may beprovided in DNA constructs. If the coding sequence and the transcripttermination region in a DNA construct are derived from differentnaturally occurring sources, the transcript termination region typicallycontains at least about 0.5 kb, preferably about 1-3 kb of sequence 3′to the structural gene from which the termination region is derived.

DNA constructs also can contain sequences encoding other polypeptides.Such polypeptides can, for example, facilitate the introduction ormaintenance of the nucleic acid construct in a host organism. Potentialhost cells include both prokaryotic and eukaryotic cells. A host cellmay be unicellular or found in a multicellular differentiated orundifferentiated organism depending upon the intended use. Dependingupon the host, regulatory elements can include elements from viral,plasmid or chromosomal genes, or the like. For expression in prokaryoticor eukaryotic microorganisms, particularly unicellular hosts, a widevariety of constitutive or inducible promoters may be employed.Expression in a microorganism can provide a ready source of a desiredpolypeptide. Among transcriptional initiation regions which have beendescribed are regions from bacterial and yeast hosts, such asEscherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, includinggenes such as beta-galactosidase, T7 polymerase, tryptophan E and thelike.

DNA constructs can also include sequences encoding other polypeptidesthat can affect the expression, activity, biochemical activity orphysiological activity of a hydroxylase or epoxygenase polypeptide. Forexample, a DNA construct can include a nucleic acid encoding a PDAT,DAGAT, lipase, FAD2 or FAD3 polypeptide, operably linked to at least oneregulatory element that confers expression in vegetative tissues or atleast one tissue of seeds of a plant. In some embodiments, a DNAconstruct includes a nucleic acid that encodes a PDAT polypeptide and anucleic acid that encodes a FAD2 polypeptide. Alternatively, such otherpolypeptide coding sequences can be provided on a separate DNAconstruct(s).

Suitable phospholipid:diacylglycerol acyltransferase (PDAT) polypeptidesand diacylglycerol acyltransferase (DAGAT) polypeptides include A.thaliana DAGAT or C. elegans DAGAT. Coding sequences for suitable PDATand DAGAT polypeptides include GenBank® Accession Nos. AAF19262,AAF19345, AAF82410 and P40345.

DAGAT and PDAT enzymes are important determinants of both the amounts(Bouvier-Nave et al. (2000) Biochem. Soc. Trans. 28(6):692-695; Jako etal. (2001) 126(2):861-874) and types (Banas et al. (2000) Biochem. Soc.Trans. 28(6):703-705; Dahlqvist et al. (2000) Proc. Natl. Acad. Sci USA,97(12):6487-6492) of fatty acids found in the triacylglycerol (TAG)fraction. Furthermore, the triacylglycerol (TAG) fraction is thepredominant repository of novel fatty acids like ricinoleic acid andvernolic acid in seeds and it is thought that this minimizes thedisruptive effects of these unusual fatty acids on plant cell membranes(Millar et al. (2000) Trends Plant Sci. 5(3):95-101). In most plants,roots, leaves, and other non-seed tissues are not usually sites of majortriacylglycerol accumulation. It is therefore likely that in non-seedtissues the activity of key enzymes in the TAG synthesis pathway such asPDATs and DAGATs are suboptimal for the contemplated application and canbe improved by overexpression of these enzymes which can result insignificant enhancement of fatty acid accumulation in the TAG fraction(Bouvier-Nave et al. (2000) Eur. J. Biochem. 267(1):85-96).

A DNA construct that encodes one or more desaturases includes constructsthat encode delta-12 fatty acid desaturases or delta-15 fatty aciddesaturases. For example, an Arabidopsis thaliana FAD2 or an Arabidopsisthaliana FAD3 polypeptide can be operably linked to a suitable promoterthat confers expression in non-seed tissues such as roots and/or leaves.The expression of a delta-12 desaturase and an epoxygenases can beuseful, since linoleic acid, the product of the desaturase, is thesubstrate converted to vernolic acid by the epoxygenase.

Nucleic acids described herein can be used to identify homologous planthydroxylase or epoxygenase coding sequences and the resulting sequencesmay provide further plant hydroxylases or epoxygenases. In particular,PCR may be a useful technique to obtain related nucleic acids fromsequence data provided herein. One skilled in the art will be able todesign oligonucleotide probes based upon sequence comparisons or regionsof typically highly conserved sequence. Of special interest arepolymerase chain reaction primers based on the conserved regions ofamino acid sequence between the hydroxylases and epoxygenases in FIG. 2(SEQ ID NOs: 13 to 24 and 34 to 42). Details relating to the design andmethods for a PCR reaction using these probes are described more fullyin the examples. If nucleic acid probes are used, they can be shorterthan the entire coding sequence. Oligonucleotides may be used, forexample, that are 10, 15, 20, or 25 nucleotides or more in length.

Hydroxylated fatty acids are found in large quantities in some naturalplant species, which suggests several possibilities for plant enzymesources. For example, hydroxy fatty acids related to ricinoleate occurin major amounts in seed oils from various Lesquerella species. Ofparticular interest, lesquerolic acid is a 20-carbon homolog ofricinoleate with two additional carbons at the carboxyl end of thechain. Other natural plant sources of hydroxylated fatty acids includeseeds of the Linum genus, seeds of Wrightia species, Lycopodium species,Strophanthus species, Convolvulaces species, Calendula species and manyothers (van de Loo et al. (1993). For example, Lesquerella densipilacontains a diunsaturated 18 carbon fatty acid with a hydroxyl group (vande Loo et al. (1993) Lipid Metabolism in Plants CRC Press, Boca Raton,p. 99-126) that is thought to be produced by an enzyme that is closelyrelated to the castor and Lesquerella fendleri hydroxylases. Similarly,epoxygenated fatty acids are found in a variety of plants includingVernonia genus, Crepis genus, Euphorbia genus and Stokesia laevis.

In addition, nucleic acids encoding a polypeptide modified from anaturally occurring sequence can be made by mutagenesis. A delta-12desaturase can for example be converted to an oleate hydroxylase bytargeted mutagenesis (Broun et al. (1998) Science, 282(5392):1315-1317;Broadwater et al. (2002) J Biol Chem. 277(18):15613-15620.). Similarchanges in coding sequences such as delta-15 (omega-3) desaturases canbe carried out to produce novel hydroxylases. As is well known in theart, once a cDNA clone encoding a plant hydroxylase or epoxygenase isobtained, it may be used to obtain its corresponding genomic nucleicacid. Thus, one skilled in the art will recognize that antibodypreparations, nucleic acid probes and the like may be prepared and usedto screen and recover homologous or related hydroxylases andepoxygenases from a variety of sources.

Typically, a nucleic acid of the invention has 70% or greater sequenceidentity, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greatersequence identity to a target nucleic acid. Sequence identity isdetermined as described herein. In some embodiments, nucleic acids arefrom 20 to 30 nucleotides, or 20 to 50 nucleotides, or 25 to 100nucleotides, or 500 to 1500 nucleotides, or 900 to 2,000 nucleotides inlength. Specific embodiments of nucleic acids include nucleotidesequences set forth in the sequence listings. It is noted that thedegeneracy of the genetic code permits codon modification without acorresponding modification of the amino acid sequence. Thus, codons in anucleic acid can be modified if desired, which may optimize expressionof a polypeptide. For example, codons with 8% or lower percentageoccurrence in a selected plant species genome can be replaced with amore frequently occurring codon, e.g., the most frequent or second mostfrequent codon for that particular amino acid. As another alternative,one member of a contiguous pair of codons can be modified if both codonshave an occurrence of 12% or lower in known sequences of the genome of aselected plant species. Data relating to codon usage database can befound, for example, at <http://www.kazusa.or.jp/codon/>.

Codons can also be changed to remove ATTTA (i.e., AUUUA) elements whichmay contribute to mRNA instability, and codons may be changed to ablatepotential polyadenylation sites. Codons can also be modified to break upruns of five or greater contiguous nucleotides of A, G, C or T (e.g.,TTTTTT). Codons can also be modified to reduce the likelihood ofaberrant splicing. Splicing potential can be assessed and donor (GT) oracceptor (AG) splice sites ablated in order to diminish splicingpotential, using predictive algorithms such as algorithms at<http://www.cbs.dtu.dk/services/NetPGene>. In addition, codons near theN-terminus of the polypeptide can be changed to codons preferred by aselected plant species, e.g., soybean (Glycine max). It will beappreciated that one or more codon modifications, including but notlimited to the modifications discussed above can be made to a nucleicacid coding sequence. Examples of sequences that have one or more codonmodification(s) to improve plant expression and have slight changes tothe amino acid sequences relative to the wild-type sequence include SEQID NOs: 28 through 33 and 129 through 133.

A nucleic acid encoding a polypeptide can have a genomic codingsequence, a cDNA coding sequence, or an mRNA coding sequence. A cDNAcoding sequence may or may not have pre-processing sequences, such astransit or signal peptide sequences. Transit or signal peptide sequencesfacilitate the delivery of the protein to a given organelle and arefrequently cleaved from the polypeptide upon entry into the organelle,releasing the “mature” sequence. The use of the precursor DNA sequencecan be useful in plant cell expression cassettes.

Transgenic Plants

According to another aspect of the invention, transgenic plants areprovided. Such plants typically express the polypeptide coding sequenceof a DNA construct described herein, resulting in an increase in theamount of a hydroxylated or epoxygenated fatty acid in vegetative planttissues or at least one tissue of seeds of such plants. A plant speciesor cultivar may be transformed with a DNA construct that encodes apolypeptide from a different plant species or cultivar (e.g., soybeantransformed with a gene encoding a castor enzyme). Alternatively, aplant species or cultivar may be transformed with a DNA construct thatencodes a polypeptide from the same plant species or cultivar.

Accordingly, a method according to the invention comprises introducing aDNA construct as described herein into a plant. Techniques forintroducing exogenous nucleic acids into monocotyledonous anddicotyledonous plants are known in the art, and include, withoutlimitation, Agrobacterium-mediated transformation, liposome fusion,microinjection, viral vector-mediated transformation, infiltration,imbibition, electroporation and particle gun transformation, e.g., U.S.Pat. Nos. 5,204,253 and 6,013,863. If a cell or tissue culture is usedas the recipient tissue for transformation, plants can be regeneratedfrom transformed cultures by techniques known to those skilled in theart. Any method that provides for transformation may be employed.

Where Agrobacterium is used for plant cell transformation, a vector maybe used which may be introduced into the Agrobacterium host forhomologous recombination with the Ti- or Ri-plasmid present in theAgrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall), the latter being permissible, solong as the vir genes are present in the transformed Agrobacterium host.The armed plasmid can give a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming plant cells, the DNA construct, bordered by the T-DNAborder(s), will be inserted into a broad host spectrum vector, therebeing broad host spectrum vectors described in the literature. Commonlyused is pRK2 or derivatives thereof. Included with the expressionconstruct and the T-DNA will be one or more markers, which allow forselection of transformed Agrobacterium and transformed plant cells. Anumber of markers have been developed for use with plant cells, such asresistance to kanamycin, the aminoglycoside G418, hygromycin, or thelike.

A number of genes that confer herbicide resistance can be used asmarkers. Genes conferring resistance to a herbicide that inhibits thegrowing point or meristem can be suitable. Exemplary genes in thiscategory code for mutant ALS and AHAS enzymes as described, for example,in U.S. Pat. Nos. 5,767,366 and 5,928,937. U.S. Pat. Nos. 4,761,373 and5,013,659 are directed to plants resistant to various imidazolinone orsulfonamide herbicides. U.S. Pat. No. 4,975,374 relates to plant cellsand plants containing a gene encoding a mutant glutamine synthetase (GS)resistant to inhibition by herbicides that are known to inhibit GS, e.g.phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,162,602discloses plants resistant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides. The resistance is conferred byan altered acetyl coenzyme A carboxylase(ACCase). Genes for resistanceto glyphosate (sold under the trade name Roundup®) are also suitable.See, for example, U.S. Pat. No. 4,940,835 and U.S. Pat. No. 4,769,061.U.S. Pat. No. 5,554,798 discloses transgenic glyphosate resistant maizeplants, which resistance is conferred by an altered5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene. Genes forresistance to phosphono compounds such as glufosinate ammonium orphosphinothricin, and pyridinoxy or phenoxy propionic acids andcyclohexones are also suitable. See European application No. 0 242 246.Other suitable herbicides include those that inhibit photosynthesis,such as a triazine and a benzonitrile (nitrilase). See U.S. Pat. No.4,810,648. Other suitable herbicides include 2,2-dichloropropionic acid,sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylureaherbicides, triazolopyrimidine herbicides, s-triazine herbicides andbromoxynil. Also suitable are herbicides that confer resistance to aprotox enzyme. See, e.g., U.S. Patent Application No. 20010016956, andU.S. Pat. No. 6,084,155. The particular marker employed is not essentialto this invention, one or another marker being suitable depending on theparticular host and the manner of construction.

Transgenic plants typically contain a DNA construct integrated intotheir genome and typically exhibit Mendelian inheritance patterns.Transgenic plants can be entered into a breeding program, e.g., tointroduce a nucleic acid encoding a polypeptide into other lines, totransfer the nucleic acid to other species or for further selection ofother desirable traits. Alternatively, transgenic plants can bepropagated vegetatively for those species amenable to such techniques.Progeny includes descendants of a particular plant or plant line.Progeny of an instant plant include seeds formed on F₁, F₂, F₃, andsubsequent generation plants, or seeds formed on BC₁, BC₂, BC₃, andsubsequent generation plants. Seeds produced by a transgenic plant canbe grown and then selfed (or outcrossed and selfed) to obtain seedshomozygous for the nucleic acid encoding a novel polypeptide.

Plants which may be employed in practicing the present inventioninclude, but are not limited to, tobacco (Nicotiana tabacum), potato(Solanum tuberosum), soybean (glycine max), peanuts (Arachis hypogaea),cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya(Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), corn (Zea mays),wheat, oats, rye, barley, rice, vegetables, ornamentals, and conifers.Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherima), and chrysanthemum. Conifers which may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Suitable grasses include Kentucky bluegrass (Poa pratensis) and creepingbentgrass (Agrostris palustris).

It is understood that hydroxylated or epoxygenated fatty acids producedby a polypeptide of the invention in planta may be subject to furtherenzymatic modification by other enzymes which are normally present in aplant or are introduced by genetic engineering methods into a plant. Forexample, lesquerolic acid, which is present in many Lesquerella species,is thought to be produced by elongation of ricinoleic acid (Moon et al.(2001) Plant Physiol. 127(4):1635-1643). Thus, the presence of a Ricinuscommunis hydroxylase construct in a transgenic plant may be sufficientto produce lesquerolic acid in the same plant, via production ofricinoleic acid by the hydroxylase polypeptide and elongation ofricinoleic acid by an endogenous polypeptide.

Nematode Resistance

Transgenic plants may be tested for hydroxy- and epoxy-fatty acidproduction in non-seed tissues. Such plants may also be tested fornematicidal activity. Similar tests for hydroxylated and epoxygenatedfatty acid production and nematicidal activity may be carried out onhairy root cultures formed by transformation with A. rhizogenes.Accordingly, the invention features a method of screening a transgenicplant for anthelmintic activity, comprising contacting the plant with anematode under conditions effective to determine whether or not theplant has anthelmintic activity. The transgenic plant has a nucleic acidencoding a hydroxylase or epoxygenase polypeptide described herein.Suitable conditions for determining anthelmintic activity are describedherein. The method can also be carried out with plant tissue, e.g., roottissue, leaf tissue or stem tissue from a transgenic plant.

In another aspect, the invention features a method for making a planthaving anthelmintic activity. As discussed herein, techniques forintroducing exogenous nucleic acids into monocotyledonous anddicotyledonous plants are known in the art. In some embodiments, forexample, a method of making a plant having anthelmintic activitycomprises (1) transforming regenerable cells of a plant species with aDNA construct described herein; and (2) regenerating one or moretransgenic plants from the cells. The resulting transgenic plant canhave a statistically significant increase in the amount of hydroxylatedor epoxygenated fatty acid in non-seed tissues compared to acorresponding untransformed counterpart. The increased level of hydroxy-or epoxy-fatty acids can result in plants that have anthelminticactivity. Nematodes that parasitize plant roots, stems, bulbs, or leavescan be controlled using the method of this invention.

As used herein, a fatty acid compound has anthelmintic activity when,tested in planta, the compound has a statistically significant increasein nematode-killing activity, a statistically significant reduction innematode fertility, a statistically significant increase in nematodesterility, a statistically significant reduction in the ability of anematode to infect or reproduce in its host, a statistically significantreduction in nematode growth or development, relative to a controltreatment in the absence of the compound. A compound having anthelminticactivity can, for example, reduce the survival time of adult nematodesrelative to unexposed similarly staged adults, e.g., by about 20%, 40%,60%, 80%, or more. In some embodiments, a compound having anthelminticactivity may also cause the nematodes to cease replicating,regenerating, and/or producing viable progeny, e.g., by about 20%, 40%,60%, 80%, or more, compared to a control treatment in the absence of thecompound.

A compound having anthelmintic activity can result in a statisticallysignificant increase in nematode repellant properties relative to acontrol treatment in the absence of the compound. In the assay, thecompound is combined with nematodes, e.g., in a well of microtiter dish,in liquid or solid media or in the soil containing the compound. Stagedadult nematodes are placed on the media. The time of survival, viabilityof offspring, and/or the movement of the nematodes are measured.

Exemplary plants-parasitic nematodes from which plants may be protectedby the present invention, and their corresponding plants, are asfollows: alfalfa: Ditylenchus dipsaci, Meloidogyne hapla, Meloidogyneincognita, Meloidogynejavanica, Pratylenchus spp., Paratylenchus spp.,Xiphinema spp.; banana: Radopholus similis, Helicotylenchusmulticinctus, Meloidogyne incognita, M. arenaria, M. javanica,Pratylenchus coffeae, Rotylenchulus reniformis; beans and peas:Meloidogyne spp., Heterodera spp., Belonolaimus spp., Helicotylenchusspp., Rotylenchulus reniformis, Paratrichodorus anemones, Trichodorusspp.; cassaya: Rotylenchulus reniformis, Meloidogyne spp.; cereals:Anguina tritici (Emmer, rye, spelt wheat), Bidera avenae (oat, wheat),Ditylenchus dipsaci (rye, oat), Subanguina radicicola (oat, barley,wheat, rye), Meloidogyne naasi (barley, wheat, rye), Pratylenchus spp.(oat, wheat, barley, rye), Paratylenchus spp. (wheat), Tylenchorhynchusspp. (wheat, oat); chickpea: Heterodera cajani, Rotylenchulusreniformis, Hoplolaimus seinhorsti, Meloidogyne spp., Pratylenchus spp.;citrus: Tylenchulus semipenetrans, Radopholus similis, Radopholuscitrophilus (Florida only), Hemicycliophora arenaria, Pratylenchus spp.,Meloidogyne spp., Bolonolaimus longicaudatus (Florida only),Trichodorus, Paratrichodorus, Xiphinema spp.; clover: Meloidogyne spp.,Heterodera trifolii; coconut: Rhadinaphelenchus cocophilus; coffee:Meloidogyne incognita (most important in Brazil), Meloidogyne exigua(widespread), Pratylenchus coffeae, Pratylenchus brachyurus, Radopholussimilis, Rotylenchulus reniformis, Helicotylenchus spp.; corn:Pratylenchus spp., Paratrichodorus minor, Longidorus spp., Hoplolaimuscolumbus; cotton: Meloidogyne incognita, Belonolaimus longicaudatus,Rotylenchulus reniformis, Hoplolaimus galeatus, Pratylenchus spp.,Tylenchorhynchus spp., Paratrichodorus minor; grapes: Xiphinema spp.,Pratylenchus vulnus, Meloidogyne spp., Tylenchulus semipenetrans,Rotylenchulus reniformis; grasses: Pratylenchus spp., Longidorus spp.,Paratrichodorus christiei, Xiphinema spp., Ditylenchus spp.; peanut:Pratylenchus spp., Meloidogyne hapla., Meloidogyne arenaria,Criconemella spp., Belonolaimus longicaudatus (in Eastern UnitedStates); pigeon pea: Heterodera cajani, Rotylenchulus reniformis,Hoplolaimus seinhorsti, Meloidogyne spp., Pratylenchus spp.; pineapple:Paratrichodorus christiei, Criconemella spp., Meloidogyne spp.,Rotylenchulus reniformis, Helicotylenchus spp., Pratylenchus spp.,Paratylenchus spp.; potato: Globodera rostochiensis, Globodera pallida,Meloidogyne spp., Pratylenchus spp., Trichodorus primitivus, Ditylenchusspp., Paratrichodorus spp., Nacoabbus aberrans; rice: Aphelenchiodesbesseyi, Ditylenchus angustus, Hirchmanniella spp., Heterodera oryzae,Meloidogyne spp.; small fruits: Meloidogyne spp.; Pratylenchus spp.,Xiphinema spp., Longidorus spp., Paratrichodorus christiei,Aphelenchoides spp. (strawberry); soybean: Heterodera glycines,Meloidogyne incognita, Meloidogyne javanica, Belonolaimus spp.,Hoplolaimus columbus; sugar beet: Heterodera schachtii, Ditylenchusdipsaci, Meloidogyne spp., Nacobbus aberrans, Trichodorus spp.,Longidorus spp., Paratrichodorus spp.; sugar cane: Meloidogyne spp.,Pratylenchus spp., Radopholus spp., Heterodera spp., Hoplolaimus spp.,Helicotylenchus spp., Scutellonema spp., Belonolaimus spp.,Tylenchorhynchus spp., Xiphinema spp., Longidorus spp., Paratrichodorusspp.; tea: Meloidogyne spp., Pratylenchus spp., Radopholus similis,Hemicriconemoides kanayaensis, Helicotylenchus spp., Paratylenchuscurvitatus; tobacco: Meloidogyne spp., Pratylenchus spp.,Tylenchorhynchus claytoni, Globodera tabacum, Trichodorus spp.,Xiphinema americanum, Ditylenchus dipsaci (Europe only), Paratrichodorusspp.; tomato: Pratylenchus spp., Meloidogyne spp.; tree fruits:Pratylenchus spp. (apple, pear, stone fruits), Paratylenchus spp.(apple, pear), Xiphinema spp. (pear, cherry, peach), Cacopaurus pestis(walnut), Meloidogyne spp. (stone fruits, apple, etc.), Longidorus spp.(cherry), Criconemella spp. (peach), and Tylenchulus spp. (olive).

Transgenic plants described herein can provide an effective,environmentally safe means of inhibiting nematode metabolism, growth,viability, fecundity, development, infectivity and/or the nematodelife-cycle. The plants may be used alone or in combination with chemicalnematicides or as part of an integrated pest management strategy.Transgenic plants can afford season-long nematode control and therebyprovide labor savings, by reducing the need for and frequency ofchemical control.

Described below are experiments demonstrating that delta-12 fatty aciddesaturase activity is essential for nematode viability. Also describedare certain nematicidal fatty acids and analogs, including nematicidalfatty acids and esters that have activity consistent with that ofdelta-12 fatty acid desaturase inhibitors. The cloning, modification,introduction into plants and expression in non-seed tissues (e.g.,roots) of DNA sequences encoding enzymes that produce these fatty acidsis also described, as are tests of regenerated plant cells, roots andplants. The following examples are to be construed as merelyillustrative, and not limiting in any way whatsoever.

EXAMPLE 1 RNA Mediated Interference RNAi

A double stranded RNA (dsRNA) molecule can be used to inactivate adelta-12 fatty acid desaturase (delta-12 fat2) gene in a cell by aprocess known as RNA mediated-interference (Fire et al. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a delta-12 fat2 nucleicacid (preferably exonic) or a fragment thereof. The dsRNA molecule canbe delivered to nematodes via direct injection, or by soaking nematodesin aqueous solution containing concentrated dsRNA, or by raisingbacteriovorous nematodes on E. coli genetically engineered to producethe dsRNA molecule.

RNAi by injection: To examine the effect of inhibiting delta-12 fat2activity, a dsRNA corresponding to the C. elegans delta-12 fat2 gene wasinjected into the nematode, basically as described in Mello et al.(1991) EMBO J. 10:3959-3970. Briefly, a plasmid was constructed thatcontains a portion of the C. elegans delta-12 fat2 sequence,specifically a fragment 651 nucleotides long, containing the entirefirst exon and terminating just before the conserved intron splicejunction between the first exon and first intron. This construct encodesapproximately the first 217 amino acids of the C. elegans delta-12 fat2gene. Primers were used to specifically amplify this sequence as alinear dsDNA. Single-stranded RNAs were transcribed from these fragmentsusing T7 RNA polymerase and SP6 RNA polymerase (the RNAs correspond tothe sense and antisense RNA strands). RNA was precipitated andresuspended in RNAse free water. For annealing of ssRNAs to form dsRNAs,ssRNAs were combined, heated to 95° C. for two minutes then allowed tocool from 70° C. to room temperature over 1.5-2.5 hours.

DsRNA was injected into the body cavity of 15-20 young adult C. eleganshermaphrodites. Worms were immobilized on an agarose pad and typicallyinjected at a concentration of 1 mg/mL. Injections were performed withvisual observation using a Zeiss Axiovert compound microscope equippedwith 10× and 40× DIC objectives, for example. Needles for microinjectionwere prepared using a Narishige needle puller, stage micromanipulator(Leitz) and an N2-powered injector (Narishige) set at 10-20 p.s.i. Afterinjection, 200 μl of recovery buffer (0.1% salmon sperm DNA, 4% glucose,2.4 mM KCl, 66 mM NaCl, 3 mM CaCl2, 3 mM HEPES, pH 7.2) were added tothe agarose pad and the worms were allowed to recover on the agarose padfor 0.5-4 hours. After recovery, the worms were transferred to NGM agarplates seeded with a lawn of E. coli strain OP50 as a food source. Thefollowing day and for 3 successive days thereafter, 7 individual healthyinjected worms were transferred to new NGM plates seeded with OP50. Thenumber of eggs laid per worm per day and the number of those eggs thathatched and reached fertile adulthood were determined. As a control,Green Fluorescent Protein (GFP) dsRNA was produced and injected usingsimilar methods. GFP is a commonly used reporter gene originallyisolated from jellyfish and is widely used in both prokaryotic andeukaryotic systems. The GFP gene is not present in the wild-type C.elegans genome and, therefore, GFP dsRNA does not trigger an RNAiphenotype in wild-type C. elegans. The C. elegans delta-12 fat2 RNAiinjection phenotype presented as a strongly reduced F1 hatch-rate, withthe few surviving individuals arrested in an early larval stage.

RNAi by feeding: C. elegans can be grown on lawns of E. coli geneticallyengineered to produce double stranded RNA (dsRNA) designed to inhibitdelta-12 fat2 expression. Briefly, E. coli were transformed with agenomic fragment of a portion of the C. elegans fat2 gene sequence,specifically a fragment 651 nucleotides long, containing the entirefirst exon and terminating just before the conserved intron splicejunction between the first exon and first intron. This construct encodesapproximately the first 217 amino acids of the C. elegans delta-12 fat2gene. The 651 nucleotide genomic fragment was cloned into an E. coliexpression vector between opposing T7 polymerase promoters. The clonewas then transformed into a strain of E. coli that carries anIPTG-inducible T7 polymerase. As a control, E. coli was transformed witha gene encoding the Green Fluorescent Protein (GFP). Feeding RNAi wasinitiated from C. elegans eggs or from C. elegans L4s. When feeding RNAiwas started from C. elegans eggs at 23° C. on NGM plates containing IPTGand E. coli expressing the C. elegans delta-12 fat2 or GFP dsRNA, the C.elegans delta-12 fat2 RNAi feeding phenotype presented as partiallysterile F1 individuals and dead F2 embryos. When feeding RNAi wasstarted from C. elegans L4 larvae at 23° C. on NGM plates containingIPTG and E. coli expressing the C. elegans DELTA-12 fat2 or GFP dsRNA,the C. elegans RNAi feeding phenotype presented as partially sterile P0individuals (i.e., the individuals exposed initially) withdevelopmentally arrested, sterile F1 nematodes. The sequence of the fat2gene is of sufficiently high complexity (i.e., unique) such that theRNAi is not likely to represent cross reactivity with other genes.

C. elegans cultures grown in the presence of E. coli expressing dsRNAand those injected with dsRNA from the delta-12 fat2 gene were stronglyimpaired indicating that the fatty acid desaturase-like gene provides anessential function in nematodes and that dsRNA from the fatty aciddesaturase-like gene is lethal when ingested by or injected into C.elegans.

EXAMPLE 2 Rescue of C. elegans Delta-12 fat2 RNAi Feeding Phenotype byLinoleic Acid Methyl Ester

The C. elegans delta-12 fatty acid desaturase (FAT-2 protein) convertsthe mono-unsaturated oleic acid to the di-unsaturated fatty acidlinoleic acid. The delta-12 fat2 RNAi prevents expression of thedelta-12 fatty acid desaturase, which is predicted to cause a decreasein levels of linoleic acid in the nematode, leading to arresteddevelopment and death. Addition of 3 mM linoleic acid methyl ester tothe NGM media used for the RNAi experiment brings about a partial rescueof the delta-12 fat2 RNAi feeding phenotype. Addition of 3 mM oleic acidmethyl ester does not rescue the delta-12 fat2 RNAi feeding phenotype(see Table 1 below).

TABLE 1 C. elegans delta-12 fat2 RNAi feeding phenotypes (starting withC. elegans L4 larvae as the P0 animal) Fatty Acid F2 Added P0 phenotypeF1 phenotype phenotype None Reduced egg laying Developmentally NA(partial sterility) arrested and sterile Oleic Acid Reduced egg layingDevelopmentally NA Methyl Ester (partial sterility) arrested and sterileLinoleic Acid Reduced egg laying Moderately delayed Slightly MethylEster development and delayed moderately reduced development egg laying

EXAMPLE 3 Preparation of Caenorhabditis elegans and Fatty Acids

Mixed stage C. elegans were washed off plates seeded with OP50 bacteriausing M9 solution. 250 μl of the M9 solution, which contained about50-100 worms, was pipetted into each well of a 24-well plate.

With the exceptions of the fatty acid salts and the free acid ofricinelaidic acid, all other fatty acid emulsions were preparedfollowing the teachings of Kim et al (U.S. Pat. No. 5,698,592). Briefly,1 mL 1% stock solution emulsions were prepared by mixing 10 μl of fattyacid with 20 μl of the surfactant Igepal CO 630 in a 1.5 mL eppendorftube. After careful mixing of fatty acid and Igepal CO 630, 850 μl ofddH₂O was added and mixed by gentle pipetting until a homogeneoussolution was obtained. Finally, 120 μl of pure isopropanol was added andmixed by gentle pipetting. 1% stock emulsions were also prepared for thepotassium salt of ricinoleic acid, the sodium salt of ricinelaidic acid,and ricinelaidic free acid. For the potassium salt of ricinoleic acid,0.01 grams were dissolved in 100 μl of ddH₂O, and combined with 20 μl ofthe surfactant Igepal CO 630 in a 1.5 mL eppendorf tube. After carefulmixing of fatty acid and Igepal CO 630, 760 μl of ddH₂O was added andmixed by gentle pipetting until a homogeneous solution was obtained.Finally, 120 μl of pure isopropanol was added and mixed by gentlepipetting. For the sodium salt and free acid of ricinelaidic acid, 0.01grams were dissolved in 100 μl of acetone, and combined with 20 μl ofthe surfactant Igepal CO 630 in a 1.5 mL eppendorf tube. After carefulmixing of fatty acid and Igepal CO 630, 760 μl of ddH₂O was added andmixed by gentle pipetting until a homogeneous solution was obtained.Finally, 120 μl of pure isopropanol was added and mixed by gentlepipetting. These stock solutions were then used to produce various fattyacid dilution emulsions in 24-well plate assays. An “acetone control”emulsion was prepared by combining 100 μl of acetone, 20 μl of thesurfactant Igepal CO 630, 760 μl of ddH₂O, and 120 μl of pureisopropanol in a 1.5 mL eppendorf tube and mixing to homogeneity.

EXAMPLE 4 Nematicidal Activity of Single Fatty Acid Methyl EsterEmulsions Against Caenorhabditis elegans

To each well, fatty acid emulsions or control emulsions were added andrapidly mixed by swirling. Nematode viability was scored by visualobservation and motility assays at various time points 24 hoursfollowing addition of emulsions or controls. The fatty acid emulsionstested were methyl esters of nonanoic (pelargonic) acid, ricinoleicacid, vernolic acid, linoleic acid, oleic acid, and control emulsionslacking fatty acids.

The structures of ricinoleic acid methyl ester, ricinelaidic acid methylester (not included in this table) and vernolic acid methyl ester aredepicted in FIG. 1.

TABLE 2 Nematicidal activity of fatty acid methyl ester emulsionsagainst C. elegans Percentage of Worm Death Fatty Acid Concentration 1hr 6 hr 24 hr Nonanoic  0.1% 100%  100%  100%  (C9-methyl ester) 0.003%50% 50% 50% Ricinoleic Acid  0.1% 80% 80% 90% (C18-methyl ester) 0.003%40% 40% 40% Vernolic Acid  0.1% 65% 65% 75% (C18-methyl ester) 0.003%20% 20% 20% Linoleic Acid  0.1% 0-5% 0-5% 0-5% (C18-methyl ester) 0.003%0-5% 0-5% 0-5% Oleic Acid  0.1% 0-5% 0-5% 0-5% (C18-methyl ester) 0.003%0-5% 0-5% 0-5% Control  0.1% 0-5% 0-5% 0-5% (no methyl ester) 0.003%0-5% 0-5% 0-5%

Both nonanoic and ricinoleic acid methyl ester emulsions are stronglynematicidal at a concentration of 0.1%. Nonanoic methyl ester emulsionscause an almost immediate cessation of nematode movement and subsequentdeath whereas ricinoleic methyl ester emulsions require up to 30 minutesbefore strong killing effects are apparent. However, at 0.003%, nonanoicacid methyl ester emulsions temporarily “stunned” C. elegans, initiallygiving the appearance of a 100% death phenotype. Several hours postinoculation, many nematodes recover and start moving again. This “stun”effect was not observed with the other fatty acid emulsions.

EXAMPLE 5 Nematicidal Activity of Single Fatty Acid Methyl Ester, Saltand Free Fatty Acid Emulsions Against Caenorhabditis elegans N2s andDauers

L: linoleic acid, R: ricinoleic acid, Re: ricinelaidic; V-trans:(12,13)-epoxy-trans-9-octadecenoic acid; ME: methyl ester

TABLE 3 Results vs. C. elegans (worm death) Fatty Acid 0.1% 0.01% 0.001%Castor Oil 10% <5% NA Pelargonic ME 100%  100%  30% L ME <5% <5% <5% Lfree acid 10% <5% <5% R ME 90% 40% 20% R free acid 95% 50% <5% Re ME100%  100%  80% Re free acid* 100%  98% 40% Potassium R 90% 15%  5%Sodium Re* 100%  100%  NA Acetone control 10%  5%  5%

TABLE 4 Results vs. C. elegans dauers (worm death) Fatty Acid 0.1% 0.01%0.001% Castor Oil NA NA NA Pelargonic ME NA NA NA L ME 40% 20% NA L freeacid 50% 40% NA R ME 70% 30% NA R free acid 90% 75% NA Re ME 100%  100% NA Re free acid* 75% 75% NA Potassium R 75% 20% NA Sodium Re* NA NA NAAcetone control 35% 20% NA V-trans ME 90% 50% NA

EXAMPLE 6 Preparation of Root Knot Nematode J2 Larvae Meloidogyne spp

M. incognita and M. javanica were prepared from tomato roots. The rootswere bleached and the debris was separated from the J2 larvae and eggsby filtration followed by sucrose density gradient centrifugation. Eggswere hatched over 4 days at 15° C. and the J2 larvae were collected bypassage though a filter, followed by centrifugation.

EXAMPLE 7 Nematicidal Activity of Fatty Acid Methyl Ester EmulsionsAgainst Root Knot Nematodes Meloidogyne spp

Nematodes and emulsions were incubated with shaking at room temperaturefor 48 hours. The contents of each well were transferred to a small spoton individual NGM plates lacking bacteria. About 24 hours after thetransfer to plates, worms on and off the inoculation spot were countedas not viable or viable, respectively. Worms were considered viable ifthey had crawled away from the inoculation spot, or if they were moving.Worms were considered non-viable if they remained at the inoculationspot.

TABLE 5 Nematicidal activity of fatty acid methyl ester emulsionsagainst M. javanica and M. incognita Fatty acid M. javanica M. incognita(0.1%) (% not viable) (% not viable) Vernolic Acid 90% 100%  (C18-methylester) Nonanoic 100%  100%  (C9-methyl ester) Ricinoleic Acid 60% 95%(C18-methyl ester) Oleic Acid 20% 25% (C18-methyl ester)

Nonanoic, vernolic and ricinoleic acid methyl ester emulsions havesignificant nematicidal activity against root knot nematodes(Meloidogyne spp.) at a concentration of 0.1%.

EXAMPLE 8 Phytotoxicity Evaluations of Fatty Acid Methyl Esters

Sterilized tomato seeds were germinated in magenta jars containingGamborg's agar media. After two weeks of growth, seedlings were treatedwith 250 μl of 1% fatty acid methyl ester emulsion (nonanoic acid,ricinoleic acid, ricinelaidic acid, oleic acid, or a control emulsionlacking any fatty acid), applied directly to the stem-media interface.Tomato seedlings were scored at various times after application ofemulsions. Of the fatty acids tested, only 1% nonanoic acid methyl esteremulsion showed obvious phytotoxic effects on the tomatoes. Within 18hours of nonanoic acid emulsion application, those tomatoes showed adistinct loss of turgor pressure (wilting phenotype) and had becomenoticeably less green in appearance. Within 24 hours, nonanoic acidtreated tomatoes were almost entirely bleached to a pale white color andhad nearly totally collapsed with most leaves lying directly on the agarmedia surface. Importantly, none of the tomatoes treated with the otherfatty acid methyl ester emulsions showed visible effects. Therefore,ricinoleic and ricinelaidic acid methyl esters show excellent potentialas anthelmintic chemicals based on their combination of high nematicidalproperties and with favorable low phytotoxicity.

EXAMPLE 9 Nematicidal Activity of Single Fatty Acid Methyl EsterEmulsions Against a Spectrum of Free-Living Animal Parasitic and PlantParasitic Nematodes

Briefly, the indicated fatty acid emulsions were added to nematodes inwells of a 24-well plate and rapidly mixed by swirling. Nematodeviability was scored by visual observation and motility assays 24 hoursfollowing addition of emulsions (48 hours for plant parasitic nematodesMeloidogyne and Heterodera species). The fatty acid emulsions testedwere methyl esters of nonanoic (pelargonic) acid, ricinelaidic acid,ricinoleic acid, vernolic acid, linoleic acid, and oleic acid. Resultsfor fatty acid emulsions against free-living, animal parasitic, andplant parasitic nematodes are combined in one table to facilitatecomparison of different emulsion activities against nematodes exhibitingdiverse lifestyles. Results shown are mean % values obtained frommultiple independent experiments

TABLE 6 Nematicidal activity of various fatty acid methyl esters againstvarious free-living, animal parasitic, and plant parasitic nematodes %Worm Death (24 hr) −control Inhibitors +control Worm (% solution) OleicLinoleic Vernolic Ricinoleic Ricinelaidic Nonanoic C. elegans (0.1%) <10<10 80 90 100 100 C. elegans (0.01%) <10 <10 50 50 100 100 C. elegans(0.001%) <10 30 30 75 30 P. trichosuri (0.1%) ~10 ~25 ~95 ~50 100 P.trichosuri (0.01%) ~10 ~25 ~90 ~60 100 P. trichosuri (0.001%) M.incognita (0.1%) 20 98 95 ~99 100 M. incognita (0.01%) 20 73 83 ~99 M.incognita (0.001%) 97 M. javanica (0.1%) 20 90 60 100 100 M. javanica(0.01%) 0-5 60 5 100 M. javanica (0.001%) ~60 H. glycines (0.1%) <10 <2030 ~60 100 100 H. glycines (0.01%) <10 <20 20 ~60 100 >95 H. glycines(0.001%) <10 <20 18 ~40 100 P. scribneri (0.1%) <20 <20 <20 <20 ~70 <20P. scribneri (0.01%) <20 <20 <20 <20 ~40 <20 P. scribneri (0.001%)

The Caenorhabditis elegans were mixed stage populations. Similar effectswere seen on several other free-living nematode species. TheParastrongyloides trichosuri (parasite of Australian bushtail possum)were dauer-like infective 3^(rd) stage larva. Similar effects are alsoseen against free-living stages. The Meloidogyne incognita andMeloidogyne javanica (root knot nematode) were 2^(nd) stage juveniles(dauer-like infective stage). The Heterodera glycines (soybean cystnematode) were 2^(nd) stage juveniles (dauer-like infective stage).Finally, the Pratylenchus scribneri (corn lesion nematode) were mixedstage populations.

As the data in the table above demonstrate, both ricinelaidic andricinoleic acid methyl ester emulsions are strongly nematicidal atconcentrations of 0.1% and 0.01%. Ricinelaidic acid methyl ester inparticular showed favorable nematicidal activity against a wide spectrumof divergent nematode genera.

EXAMPLE 10

The following table lists primers used in the cloning and preparation ofvarious nucleic acids constructs including hydroxylases, epoxygenases,5′-UTRs and 3′-UTRs.

TABLE 7 Sequence primers used in cloning SEQ ID Name Sequence NOHomology to Hyd1 atgggaggtggtggtcgcatg 46 first 7 codons of R. communisHyd2 ttaatacrtgttccggtacca 47 last 7 codons of R. communis Les1atgggtgctggtggaagaataatg 48 first 8 codons of L. fendleri Les10tcataacttattgaagtaatagtagacaccttt 49 last 11 codons of L. fendleri les6tcataacttattgttgtaata 50 last 7 codons of L. fendleri Ecrep2gcaatccctccccattg 51 codons 33-38 of C. biennis Ecrep8tcacaatttatcataccaataaacacc 52 last 9 codons of C. biennis 5′UTR-HIIIFatacaaaagcttagagagagagattctgcgga 53 first 20 nt of A. thaliana Fad25′ UTR 3′UTR-SphIR attcaatgcatgcaacataatgagcagccaaaa 54 last 20 nt of A.thaliana Fad2 3 UTR Fad-HIIIF attcaataagcttatgggtgcaggtggaagaat 55 first7 codons of A. thaliana Fad2 Fad-SphIR atacaagcatgctcataacttattgttgtacc56 last 7 codons of A. thaliana Fad2 3′Fad/casaagcaatggggtgggatggctttcttcagatctcccaccg 57 codons 31-38 Fad2/codons43-49 R. communis 5′Fad/cas cggtgggagatctgaagaaagccatcccaccccattgctt 58codons 31-47 Fad2/codons 43-49 R. communis Cas-SalRgtcgacatacttgttccggtaccaga 59 last 7 codons of R. communis 3′Fad/lescgattgctttcttcagatctcccaccgagaaaggcggtt 60 codons 28-33 Fad2/codons35-41 L. fendleri 5′Fad/les aaccgcctnctcggtgggagatctgaagaaagcaatcc 61codons 28-33 Fad2/codons 35-41 L. fendleri Les-SalIRgtcgactaacttattgttgtaatagt 62 last 7AA of L. fendleri 3′Fad/lindgggattgctttccttagatctcccaccgagaaaggcggtt 63 codons 28-33 Fad2/codons35-41 L. lindheimeri 5′Fad/lind aaccgcctttctcggtgggagatctaaggaaagcaatccc64 codons 28-33 Fad2/codons 35-41 L. lindheimeri Lind-SalIRgtcgactaacttattgttgtaatagt 65 last 7 codons of L. lindheimeri 3′Fad/gracaaccgccmctcggtgggagatctgaagaaagcaatccc 66 codons 28-33 Fad2/codons 35-41L. gracilis 5′Fad/grac gggattgctttcrtcagatctcccaccgagaaaggcggtt 67codons 28-33 Fad2/codons 35-41 L. gracilis Grac-SalIRgtcgactcataacttattgttgtaat 68 last 7 codons of L. gracilis 3′Fad/crepcggtgggagatctgaagaaagcaatccctccccattgctt 69 codons 32-38 Fad2/first 7codons of partial C. biennis 5′Fad/crepaagcaatggggagggartgctttcrtcagatctcccaccg 70 codons 32-38 Fad2/first 7codons of partial C. biennis clone Crep-SalIR gtcgaccaatttatgataccaataaa71 last 7 codons of partial C. biennis clone 5′CastorhindIII-katacaaaagcttataatgggaggtggtggtcgcat 72 first 7 codons of R. communis3′CastorBamHI atacaaggatccttaatacttgttccggtacc 73 last 7 codons of R.communis Castor-HANOTI atacaagcggccgcagcgtaatctggaacatcgt 74 last 7codons of R. communis 5′fendhindIII-Katacaaaagcttataatgggtgctggtggaagaat 75 first 7 codons of L. fendleri3′fendBamHI atacaaggatcctcataacttattgttgtaat 76 first 7 codons of L.fendleri 5′HindIIIK/HA/fend atacaaaagcttataatgtacccatacgatgttcc 77 first7 codons of L. fendleri UT3 atgagagctcgtttaaacgattttaatgtttagc 78 first24 nt of UBI3 term UT4 atgagaattcggccggccaatagtctcgac 79 last 20 nt ofUBI3 term UP1 tcatgaggcgcgccaaagcacatacttatcg 80 first 17 nt of UBI3promoter UP2 atgagcatgcaagcttcttcgcctggaggagag 81 last 23 nt of UBI3promoter HA5 agctatgtacccatacgatgttccagattacgctg 82 HA tag HA6tcgacagcgtaatctggaacatcgtatgggtacat 83 HA tag CHA1gatccatgtacccaatacgatgttccagattacgctctcgaggagct 84 HA tag CHA2ctcgagagcgtaatctggaacatcgtatgggtacatg 85 HA tag IRT1atgaggcgcgccctttctctgacttttaacatcc 86 first 22 nt of IRT2 promoter IRT2actggcatgcgtattgagattgttttataatatatg 87 last 26 nt of IRT2 promoterCastor 5′HindIII atacaaaagcttatgggaggtggtggtcgcat 88 first 6 codons ofR. communis Casotr 3′BamHI atacaaggatccatacttgttccggtaccaga 89 last 6codons of R. communis fend F SalI atacaaaagcttatgggtgctggtggaagaat 90first 6 codons of L. fendleri Fend R B-stopatacaaggatcctaacttattgttgtaatagt 91 last 6 codons of L. fendleri Castor5′ SalI atacaagtcgacatgggaggtggtggtcgcat 92 first 6 codons of R.communis Castor 3′ BamHI atacaaggatccatacttgttccggtaccaga 93 last 6codons of R. communis 5′ΔKKGG2 ataaccagcaacaacagtgagagcagccaccttaagcgagc94 codons 11-17, codons 22-27 of R. communis 3′ΔKKGG2gctcgcttaaggtggctgctctcactgttgttgctggttat 95 codons 11-17, codons 22-27of R. communis 5′ΔT ttcttcctcagcctctctcttacctagcttggcctctctat 96 codons76-82, codons 84-90 of L. gracilis 3′ΔTatagagaggccaagctaggtaagagagaggctgaggaagaa 97 codons 76-82, codons 84-90of L. gracilis castor XbaI MfeI R caattgtctagattaatacttgttccggtaccag 98last 22 nt of R. communis HIII NcoI castor F aagcttaccatgggaggtggtggtcg99 first 17 nt of R. communis M13 Reverse gaaacagctatgaccatg 100 M13bacteriophage (M13/pUC plasmids) gracilis XbaI MfeIcaattgtctagatcataacttattgttgtaatag 101 last 22 nt of L. gracilis R HIIINcoI gracilis aagcttaccatgggtgctggtggaagaat 102 first 20 nt of L.gracilis F Crepis XbaI MfeI R caattgtctagatcacaatttatgataccaataaa 103last 23 nt of C. biennis BamHI castor Fatacaaggatccaaatgggaggtggtggtcgcat 104 first 20 nt of R. communis BamHIgracilis F atacaaggatccaaatgggtgctggtggaagaat 105 first 20 nt of L.gracilis BamHI NcoI S. aggatccctaccatgggtgcaggtggtcggat 106 first 20 ntof S. laevis epoxygenase F S. epoxygenase tctagattacattttatggtaccagtaaa107 last 20 nt of S. laevis XbaI R BgIII NcoI C.agatctctaccatgggtgcccacggccatgg 108 first 20 nt of C. biennis biennis FHA-tag-F agcttctcgagaccatggcgtacccgtacgacgtgcccgactacgccag 109 HA tagHA-tag-R gatcctggcgtagtcgggcacgtcgtacgggtacgccatggtctcgaga 110 HA tagFad5′UTR-F atcctcgagagagattctgcggaggagcttc 111 Fad2 5′ UTR of A.thaliana Fad5′UTR-R atcggatccatggrtctgcagaaaaccaaaagca 112 Fad2 5′ UTRof A. thaliana Fad3′UTR-F atctctagatgaggatgatggtgaagaaattg 113 Fad23′ UTR of A. thaliana Fad3′UTR-R atcaagcttactgtccgaaggtcacatttc 114 Fad23′ UTR of A. thaliana Crep12F ggaatgcatgtacatcgagcc 115 codons 355-360of C. biennis Crep13R ggaacttgtgttggcatggtg 116 codons 138-144 of C.biennis Estok-14 tggccngtntaytggttytg 117 codons 81-87 of S. laevisEstok-17 tcyttngcytcyctccacat 118 codons 350-356 of S. laevis SI-1atgggtgctggtggtcggatg 119 codons 1-7 of S. laevis Stok-1Rgaacacgcttacacctaggac 120 codons 254-260 of S. laevis Stok12Ratcaatccactggtattcac 121 codons 109-114 of S. laevis Stok14Fgtcctaggtgtaagcgtg 122 codons 254-259 of S. laevis HIII NcoI C.aagcttaccatgggtgcccacggccatgg 123 first 20 nt of C. biennis biennis FAscI NcoI C. ggcgcgccaccatgggtgcccacggccatgg 124 first 20 nt of C.biennis biennis F

EXAMPLE 11

The table below lists promoters and UTRs that can be used to achieveexpression of polypeptides in plant vegetative tissue.

TABLE 8 Promoter-UTR sequences for genes strongly expressed in plantroots Element Species - Gene Accession Nucleotides TobRB7 Nicotianatabacum (common tobacco) - aquaporin S45406 1 to 1953 TUB-1 Arabidopsisthaliana (thale cress) - beta 1-tubulin M20405 1 to 569 PsMTA Pisumsativum (pea) - metallothionein-like protein Z23097 1 to 804 RPL16AArabidopsis thaliana (thale cress) - ribosomal protein X81799 1 to 1014L16 ARSK1 Arabidopsis thaliana (thale cress) - serine/threonine L22302 1to 807 protein kinase AKT1 Arabidopsis thaliana (thale cress) -potassium U06745 1 to 231 transporter LJAS2 Lotus japonicus - asparaginesynthetase X89410 1 to 144 MsH3g1 Medicago sativa - cultivar chiefhistone H3.2 U09458 1 to 482

EXAMPLE 12

This example describes the cloning of delta-12 desaturase-likehydroxylases and epoxygenases (SEQ ID NOs: 1 to 6 and 27 in the sequencelistings).

Cloning of Castor Oleate Hydroxylase Gene

Genomic DNA was isolated from Ricinus communis leaf tissue. The senseprimer Hyd1 (SEQ ID NO: 46) and antisense primer Hyd2 (SEQ ID NO: 47)were used to amplify a genomic copy of the castor hydroxylase gene in aGradient PCR reaction [30 thermal cycles (1 min 95° C., 30 sec 48-63°C., 2 min 68° C.)] with KTLA DNA polymerase under standard conditions.The PCR product was fractionated in a 1% agarose gel. Bandsapproximately 1100 bp long were excised and gel purified (QIAquick GelExtraction). DNA was cloned using a TOPO TA kit (Invitrogen). Candidateclones were sequenced in their entirety with an automated DNA sequencer(such as model 373 from Applied Biosystems, Inc.)

Cloning of Lesquerella lindheimeri and Lesquerella gracilis BifunctionalHydroxylase Genes

Genomic DNA was isolated from L. lindheimeri and L. gracilis leaftissue. Sense primer Les1 (SEQ ID NO: 48) and antisense primer Les10(SEQ ID NO: 49) were used to amplify genomic copies of both Lesquerellabifunctional hydroxylase genes in a PCR reaction [30 thermal cycles (2min 94° C., 1 min 55° C., 2 min 68° C.)] with KTLA DNA polymerase understandard conditions. The PCR product was fractionated in a 1% agarosegel. Bands approximately 1100 bp long were excised and gel purified(QIAquick Gel Extraction). DNA was cloned using a TOPO TA kit(Invitrogen). Candidate clones were sequenced in their entirety with anautomated DNA sequencer (such as model 373 from Applied Biosystems,Inc.)

Cloning of Lesquerella fendleri Bifunctional Hydroxylase Gene

Genomic DNA was isolated from L. fendleri. Sense primer Les1 (SEQ ID NO:48) and antisense primer Les6 (SEQ ID NO: 50) were used to amplify agenomic copy of the L. fendleri bifunctional hydroxylase gene in aGradient PCR reaction [30 thermal cycles (1 min 95° C., 30 sec 45-63°C., 2 min 68° C.)] with KTLA DNA polymerase under standard conditions.The PCR product was fractionated in a 1% agarose gel. Bandsapproximately 1100 bp long were excised and gel purified (QIAquick GelExtraction). DNA was cloned using a TOPO TA kit (Invitrogen). Candidateclones were sequenced in their entirety with an automated DNA sequencer(such as model 373 from Applied Biosystems, Inc.)

Cloning of Crepis biennis Epoxygenase Gene

Genomic DNA was isolated from C. biennis. The sense primer Ecrep2 (SEQID NO: 51) and antisense primer Ecrep8 (SEQ ID NO: 52) were used toamplify a partial genomic clone of the C. biennis epoxygenase gene in aGradient PCR reaction [30 thermal cycles (1 min 95° C., 30 sec 45-63°C., 2 min 68° C.) with KTLA DNA polymerase under the standardconditions]. The PCR product was fractionated on a 1% agarose gel and aband approximately 1100 bp long was excised and gel purified (QIAquickGel Extraction). The gene fragment was then cloned using a TOPO TAcloning kit (Invitrogen). Candidate clones were sequenced in theirentirety with an automated DNA sequencer (such as model 373 from AppliedBiosystems, Inc.) to yield plasmid clone, Div2966. Partial sequence datafor the C. biennis epoxygenase was obtained from Div2966, includingnucleotide sequence for codons 33-374 and the 3′ stop codon. The clonelacked the first 32 codons of the C. biennis epoxygenase, as well as the5′ untranslated region. To obtain the missing 5′ sequence of the C.biennis epoxygenase gene, the inverse PCR technique was applied. InversePCR permits the rapid amplification of unknown segments of DNA thatimmediately flank a target sequence. Briefly, C. biennis genomic DNA isdigested with a selected restriction enzyme, then ligated to circularizesmaller segments of genomic DNA. These circularized segments are thenused as templates for PCR with primers directing DNA amplificationoutward away from the known region of the gene of interest to amplifythe missing flanking sequences. Inverse PCR can be used to amplifymissing 5′ or 3′ sequences. The digested, ligated, and circularizedgenomic DNA was directly PCR amplified using gene-specific primers(Crep12F; SEQ ID NO: 115 and Crep13R; SEQ ID NO: 116) designed from theknown sequence that anneal within the gene of interest. This procedurewas performed to generate clone Div4373, which contains codons 1-137 and355-374. Taken together, clone Div2966 and Div4373 contain sequencescomprising the complete open reading frame of the epoxygenase gene of C.biennis.

Cloning of Stokesia leavis Epoxygenase Gene

Genomic DNA was isolated from S. laevis. Degenerate primers weredesigned to anneal to regions within the S. leavis epoxygenase genewhich were predicted to exhibit a high degree of sequence conservationacross many plant epoxygenases. The sense primer Estok14 (SEQ ID NO:117) and antisense primer Estok17 (SEQ ID NO: 118) were used to amplifya genomic fragment of the S. laevis epoxygenase gene. Amplified PCRproducts were then cloned into a suitable vector for DNA analysis. Thisprocedure was performed to obtain clone Div4023. This clone containedcodons 88-356. To obtain the 5′ end sequence of the gene, gene-specificprimers were designed from known sequence that anneal within the gene ofinterest, and a sense primer S1-1 (SEQ ID NO: 119) and an antisenseprimer Stok1R (SEQ ID NO: 120), were used to amplify the rest of theepoxygenase gene. This yielded plasmid clone Div4172. This clonecontained codons 1-260. To obtain the 3′ end of the S. laevisepoxygenase gene, the inverse PCR technique was applied. Inverse PCRpermits the rapid amplification of unknown segments of DNA thatimmediately flank a target sequence. Briefly, S. laevis genomic DNA isdigested with a selected restriction enzyme, then ligated to circularizesmaller segments of genomic DNA. These circularized segments are thenused as templates for PCR with primers directing DNA amplificationoutward away from the known region of the gene of interest to amplifythe missing flanking sequences. Inverse PCR can be used to amplifymissing 5′ or 3′ sequences. The digested, ligated, and circularizedgenomic DNA was directly PCR amplified using the gene-specific primersStok12R (SEQ ID NO: 121) and Stok14F (SEQ ID NO: 122), which weredesigned from the known sequence that anneal within the gene ofinterest. This procedure was performed to generate clone Div4324, whichcontains codons 1-108 and 254-377. Taken together, clone Div4023,Div4172 and Div4324 contain sequences comprising the complete openreading frame of the epoxygenase gene of S. laevis.

Cloning of ΔT L. gracilis Bifunctional Hydroxylase Construct:

Specific primers were designed to remove nucleotides 245-247 (CTA) fromthe full length R. communis hydroxylase gene. A two-round PCR basedsubcloning strategy was used to generate the ΔT L. gracilis bifunctionalhydroxylase. The first round of PCR primers were as follows; to amplify5′ end of the bifunctional hydroxylase excluding nucleotides 245-247,the sense primer M13 Reverse (SEQ ID NO: 100) and antisense primer 3′AT(SEQ ID NO: 97) were used in a PCR reaction using a copy of the L.gracilis bifunctional hydroxylase gene contained in the cloning vectorpCR2.1 as a template. To amplify the 3′ end of the bifunctionalhydroxylase gene excluding nucleotides 245-247, the sense primer 5′ΔT(SEQ ID NO: 96) and antisense primer gracilis XbaI MfeI R (SEQ ID NO:101) were used. For the second round of PCR, the sense primer HIII NcoIgracilis F (SEQ ID NO: 102) and antisense primer gracilis XbaI Mfe R(SEQ ID NO: 101) were used to generate the final PCR product ΔT L.gracilis hydroxylase. PCR products were amplified using 5 thermal cycles(1 min, 94 C, 30 sec 50° C., 1.5 min 68° C.) and then 15 thermal cycles(1 min, 94° C., 30 sec 57° C., 1.5 min 68° C.) with KTLA DNA polymeraseunder standard conditions. The construct was then subcloned into a plantexpression vector using the NcoI and XbaI restriction enzymes sites.

Cloning of the ΔKKGG Ricinus communis Hydroxylase Construct:

Specific primers were designed to remove nucleotides 53-64(AGAAAGGAGGAA, SEQ ID NO: 140) from the full length R. communishydroxylase gene. A two-round PCR based subcloning strategy was used togenerate the ΔKKGG Ricinus communis hydroxylase gene. The first round ofPCR primers were as follows; to amplify 5′ end of the Ricinushydroxylase gene excluding nucleotides 53-64, the sense primer M13Reverse (SEQ ID NO: 100) and antisense primer 3′ΔKKGG2 (SEQ ID NO: 95)were used in a PCR reaction using a copy of the R. communis hydroxylasegene contained in the cloning vector pCR2.1 as a template. To amplifythe 3′ end of the Ricinus hydroxylase gene excluding nucleotides 53-64,the sense primer 5′ΔKKGG2 (SEQ ID NO: 94) and antisense primer castorXbaI MfeI R (SEQ ID NO: 98) were used. For the second round of PCR, thesense primer HIII NcoI castor F (SEQ ID NO: 99) and castor XbaI Mfe R(SEQ ID NO: 98) were used to generate the final PCR product ΔKKGGRicinus communis hydroxylase. PCR products were amplified using 5thermal cycles (1 min, 94° C., 30 sec 50° C., 1.5 min 68° C.) and then15 thermal cycles (1 min, 94° C., 30 sec 57° C., 1.5 min 68° C.) withKTLA DNA polymerase under standard conditions. The construct was thensubcloned into a plant expression vector using the NcoI and XbaIrestriction enzymes sites.

EXAMPLE 13

This example describes the isolation of the Arabidopsis thaliana fad2regulatory and coding sequences and the construction of fad2/hydroxylaseand fad2/epoxygenase fusion polypeptides. See SEQ ID NO: 7 to 12 in thesequence listings.

Isolation of the A. thaliana fad2 Desaturase cDNA Clone

Total RNA was isolated from A. thaliana leaf tissue (Qiagen RNeasy).RT-PCR was performed using the Roche Titan One Tube RT-PCR system withthe sense primer 5′UTR-HIIIF (SEQ ID NO: 53) and antisense primer3′UTR-SphIR (SEQ ID NO: 54). RT-PCR was set up following the kitdirections [1 cycle (30 minutes 50° C.), 1 cycle (2 minutes 94° C.), 10cycles (10 seconds 94° C., 30 seconds 60° C., 1 minute 68° C.), 25cycles (10 seconds 94° C., 30 sec 60° C., 1 min 68° C.+cycle elongationof 5 seconds for each cycle), 1 cycle (7 min 68° C.)]. Bandsapproximately 1100 bp long were excised and gel purified (QIAquick GelExtraction). DNA was cloned using a TOPO TA kit (Invitrogen). Candidateclones were sequenced in their entirety with an automated DNA sequencer(Model 373 from Applied Biosystems, Inc.)

Isolation of the A. thaliana fad2 Desaturase Genomic DNA Clone

Genomic DNA was isolated from A. thaliana leaf tissue. The sense primer5′UTR-HIIIF (SEQ ID NO: 53) and antisense primer 3′UTR-SphIR (SEQ ID NO:54) were used to amplify genomic fad2 DNA in a PCR reaction [5 thermalcycles (1 min 95° C., 30 sec 54° C., 2 min 68° C.), 25 thermal cycles (1min 95° C., 30 sec 62° C., 2 min 68° C.)] with KTLA DNA polymerase underthe standard conditions. The PCR product was fractionated in a 1%agarose gel. Bands approximately 2400 bp long were excised and gelpurified (QIAquick Gel Extraction). DNA was cloned using a TOPO TA kit(Invitrogen). Candidate clones were sequenced in their entirety with anautomated DNA sequencer (such as model 373 from Applied Biosystems,Inc.)

Generation of fad2/Ricinus communis Hydroxylase Chimeric cDNA

A two-round PCR based subcloning strategy was used to generate all ofthe chimeric cDNAs. In the first round of PCR, the sense primerFad-HIIIF (SEQ ID NO: 55) and antisense primer 3′Fad/cas (SEQ ID NO: 57)were used to amplify the first 114 bases from the fad2 cDNA clone. Thesense primer 5′-Fad/cas (SEQ ID NO: 58) and antisense primer Cas-SalR(SEQ ID NO: 59) were used to amplify the last 1034 bases (excluding TAA)of the Ricinus communis hydroxylase cDNA clone by PCR [1 thermal cycle(4 min 94° C.), 5 thermal cycles (45 sec 94° C., 45 sec 50° C., 60 sec68° C.), 25 thermal cycles (45 sec 94° C., 45 sec 57° C., 60 sec 68° C.)with KTLA DNA polymerase under the standard conditions. The PCR productswere fractionated on a 1% agarose gels. The bands were excised andcleaned (QIAquick Gel Extraction—final volume 50 uL). The clean productwas diluted 1:100 (TE) and both DNAs were used as the template (1 μLeach) in the second round of PCR. In the second round of PCR senseprimer Fad-HIIIF (SEQ ID NO: 55) and antisense primer Cas-SalR (SEQ IDNO: 59) were used to generate the final PCR product fad2/Ricinuscommunis chimeric cDNA [1 thermal cycle (4 min 94° C.), 5 thermal cycles(45 sec 94° C., 45 sec 50° C., 60 sec 68° C.), 25 thermal cycles (45 sec94° C., 45 sec 57° C., 60 sec 68° C.) with KTLA DNA polymerase understandard conditions]. A band approximately 1300 bp long was excised andgel purified (QIAquick Gel Extraction). DNA was cloned using a TOPO TAkit (Invitrogen). Candidate clones were sequenced in their entirety withan automated DNA sequencer (such as model 373 from Applied Biosystems,Inc.)

Generation of fad2/Lesquerella fendleri Hydroxylase Chimeric cDNA

The same two-round PCR based subcloning strategy was used to generatethe fad2/Lesquerella fendleri chimeric cDNA. The first round PCR primerswere as follows; to amplify the 5′ end of the A. thaliana fad2, thesense primer Fad-HIIIF (SEQ ID NO: 55) and antisense primer 3′-Fad/les(SEQ ID NO: 60) were used. To amplify the 3′ end of the L. fendleribifunctional hydroxylase gene, the sense primer 5′Fad/les primer (SEQ IDNO: 61) and antisense primer Les-SalIR (SEQ ID NO: 62) were used. In thesecond round of PCR, the sense primer Fad-HIIIF (SEQ ID NO: 55) andantisense primer Les-SalIR (SEQ ID NO: 62) were used to generate thefinal PCR product fad2/Lesquerella fendleri chimeric cDNA.

Generation of fad22/Lesquerella lindheimeri Hydroxylase Chimeric cDNA

The same two-round PCR based subcloning strategy was used to generatethe fad2/Lesquerella lindheimeri chimeric cDNA. The first round of PCRprimers were as follows; to amplify the 5′ end of the A. thaliana fad2,the sense primer Fad-HIIIF (SEQ ID NO: 55) and antisense primer3′-Fad/lind (SEQ ID NO: 63) were used. To amplify the 3′ end of the L.lindheimeri bifunctional hydroxylase gene, the sense primer 5′Fad/lindprimer (SEQ ID NO: 64) and antisense primer Lind-SalIR (SEQ ID NO: 65)were used. In the second round of PCR, the sense primer Fad-HIIIF (SEQID NO: 55) and antisense primer Lind-SalIR (SEQ ID NO: 65) were used togenerate the final PCR product fad2/Lesquerella lindheimeri chimericcDNA.

Generation of fad2/Lesquerella gracilis a Hydroxylase Chimeric cDNA

The same two-round PCR based subcloning strategy was used to generatethe fad2/Lesquerella gracilis A chimeric cDNA. The first round of PCRprimers were as follows; to amplify the 5′ end of the A. thaliana fad2,the sense primer Fad-HIIIF (SEQ ID NO: 55) and antisense primer3′-Fad/grac (SEQ ID NO: 66) were used. To amplify the 3′ end of the L.gracilis bifunctional hydroxylase gene, the sense primer 5′-Fad/gracprimer (SEQ ID NO: 67) and antisense primer Grac-SalIR (SEQ ID NO: 68)were used. In the second round of PCR, the sense primer Fad-HIIIF (SEQID NO: 55) and antisense primer Grac-SalIR (SEQ ID NO: 68) were used togenerate the final PCR product fad2/Lesquerella gracilis A chimericcDNA.

Generation of fad2/Crepis biennis Epoxygenase Chimeric cDNA

The same two-round PCR based subcloning strategy was used to generatethe fad2/Crepis biennis chimeric cDNA. The first round of PCR primerswere as follows; to amplify the 5′ end of the A. thaliana fad2, thesense primer Fad-HIIIF (SEQ ID NO: 55) and antisense primer 3′-Fad/crep(SEQ ID NO: 69) were used. To amplify the 3′ end of the C. biennisepoxygenase, the sense primer 5′Fad/crep primer (SEQ ID NO: 70) andantisense primer Crep-SalIR (SEQ ID NO: 71) were used. In the secondround of PCR, the sense primer Fad-HIIIF (SEQ ID NO: 55) and antisenseprimer Crep-SalIR (SEQ ID NO: 71) were used to generate the final PCRproduct fad2/Crepis biennis chimeric cDNA.

EXAMPLE 14

This example describes the construction of eleven (11) synthetic,optimized hydroxylase and epoxygenase sequences.

Five codon-optimized hydroxylase (Ricinus communis, HA-tagged Ricinuscommunis and Lesquerella gracilis) and epoxygenase (Stokesia laevis Aand Crepis biennis) sequences were constructed as follows. First the2nd, 3^(rd), and 4th codons downstream of the initiation methioninecodon were changed to GCT, TCC, and TCC (encoding alanine, serine andserine). Secondly, codons with 8% or lower percentage occurrence ineither the Arabidopsis thaliana, Glycine max, Lycopersicon esculentum orNicotiana tabacum genomes (e.g., CGG for arginine) were replaced withthe most frequent or second most frequent codon for that particularamino acid (e.g., AGA or AGG for arginine). Finally, one member of acontiguous pair of codons was optimized if both codons had an occurrenceof 12% or lower in either the Arabidopsis thaliana, Glycine max,Lycopersicon esculentum or Nicotiana tabacum genomes. Data for the codonoptimization process were taken from the codon usage database(http://www.kazusa.or.jp/codon/).

Codons were also changed to remove ATTTA (i.e., AUUUA) elements whichmay destabilize mRNAs, to ablate potential polyadenylation sites, and tobreak up runs of A, G, C or T of five or greater nucleotides (e.g.,TTTTT). Codons were also modified to reduce the likelihood of aberrantsplicing. Splicing potential was assessed with the NetPlantGeneprediction server (http://www.cbs.dtu.dk/services/NetPGene/). Whenever adonor and acceptor existed where both were predicted with greater than0.9 confidence a codon was mutated to ablate either the donor (GT) oracceptor (AG) sites and thus diminish splicing potential. SEQ ID NOS:30, 31, 32 and 129 are examples of these optimized sequences.

Additional codon optimized variants of the Ricinus communis andLesquerella gracilis hydroxylase and Crepis biennis, Crepis palaestinaand a second Stokesia laevis (Stokesia laevis B) epoxygenase gene weremade. These additional sequences contained modifications to more closelymimic the most common soybean (Glycine max) codons. The 2nd, 3^(rd), and4th codons downstream of the initiation methionine codon were changed toGCT, TCC, and TCC (encoding alanine, serine and serine). Codons werealso changed to remove ATTTA (i.e., AUUUA) elements which maydestabilize mRNAs, to ablate potential polyadenylation sites, and tobreak up runs of A, G, C or T of five or greater nucleotides (e.g.,TTTTT). Codons were also modified to reduce the likelihood of aberrantsplicing. Splicing potential was assessed with the NetPlantGeneprediction server (http://www.cbs.dtu.dk/services/NetPGene/). Whenever adonor and acceptor existed where both were predicted with greater than0.9 confidence a codon was mutated to ablate either the donor (GT) oracceptor (AG) sites and thus diminish splicing potential. Data for codonoptimization procedures were taken from the codon usage database(http://www.kazusa.or.jp/codon/). SEQ ID Nos.: 28, 29, 130, 131, 132 and133 are examples of such optimized R. communis, S. laevis A, C.palaestina, S. laevis B, C. biennis and L. gracilis genes, respectively.

EXAMPLE 15

This example describes the expression of hydroxylase, bifunctionalhydroxylase and epoxygenase polypeptides in Saccharomyces cerevisiae andanalysis of the fatty acid profiles in yeast by GC-MS.

Yeast Stains, Media, and Culture Conditions

Saccharomyces cerevisiae strains YPH499 (MATa ura3-52 lys2-801 ase2-101trp1-Δ63 his3-Δ2000 leu2-Δ1) and INVsc1 (MATa his3-Δ1 leu2 trp1-289ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52) were used throughout thesestudies.

Plasmid for Yeast Transformation

The plasmid pYES2 (Invitrogen) was used to transform yeast strains. Theplasmid contains an E. coli replication origin, a yeast plasmidreplication origin, an E. coli ampicillin resistance gene and the yeastgene URA3. It utilizes an expression cassette including agalactose-inducible promoter (GAL-1).

Cloning Genes of Interest into Yeast Expression Vector pYES2

Modification of the R. communis hydroxylase and L. gracilis bifunctionalgenomic clones were performed by PCR amplification using specificprimers.

Ricinus communis hydroxylase: The following specific primers weredesigned to introduce a Kozak consensus sequence and a HindIIIrestriction site immediately upstream of the initiation codon and aBamH1 site immediately downstream of the stop codon: Direct primer:5′-CastorhindIII-k (SEQ ID NO: 72) and Reverse primer: 3′CastorBamHI.(SEQ ID NO: 73). The hydroxylase was amplified by PCR [5 thermal cycles(1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) and then 25 thermalcycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.) with KTLA DNApolymerase under standard conditions]. The PCR product was digested withHindIII and BamH1 and subsequently cloned into HindIII, BamH1 of pYES2yeast expression vector.

Ricinus communis hydroxylase with a C-terminal HA tag: The followingspecific primers were designed to introduce a Kozak consensus sequenceand a HindIII site immediately upstream of the start codon and a NotIsite and HA tag immediately before the stop codon: Direct primer:5′-castorhindIII-k (SEQ ID NO: 72), and the Reverse primer:5′-castor-HANOTI (SEQ ID NO: 74). The hydroxylase with a C-terminal HAtag was amplified by PCR [5 thermal cycles (1 min, 92° C., 30 sec 50°C., 1.5 min 68° C.) and then 25 thermal cycles (1 min, 92° C., 30 sec57° C., 1.5 min 68° C.) with KTLA DNA polymerase under standardconditions]. The PCR product was digested with HindIII and NotI andsubsequently cloned into the HindIII, NotI sites of the pYES2 expressionvector.

Ricinus communis hydroxylase with a N-terminal HA tag: The followingprimers were designed for construction of a Ricinus communis hydroxylasewith a N-terminal HA tag, Direct primer: BamHI castor F (SEQ ID NO:104), and Reverse primer: castor XbaI MfeI R(SEQ ID NO: 98). Thehydroxylase was amplified by PCR [5 thermal cycles (1 min, 92° C., 30sec 50° C., 1.5 min 68° C.) and then 25 thermal cycles (1 min, 92° C.,30 sec 57° C., 1.5 min 68° C.) with KTLA DNA polymerase under standardconditions]. The PCR product was digested with BamHI/MfeI and subclonedinto the BamHI/EcoRI sites of the pUC-HA vector. The hydroxylase plusthe N-terminal HA tag was then subcloned (HindIII/XbaI) into the yeastexpression vector pYES2.

Lesquerella lindheimeri bifunctional enzyme: The following specificprimers were designed to introduce a Kozak consensus sequence and aHindIII restriction site immediately upstream of the initiation codonand a BamH1 site immediately downstream of the stop codon: Directprimer: 5′-fendhindIII-K (SEQ ID NO: 75), and Reverse primer:3′-fendBamHI (SEQ ID NO: 76). The hydroxylase was amplified by PCR [5thermal cycles (1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) and then25 thermal cycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.) withKTLA DNA polymerase under standard conditions]. The PCR product wasdigested with HindIII, BamH1 and cloned into the HindIII, BamH1 of pYES2yeast expression vector.

Lesquerella lindheimeri bifunctional enzyme with a N-terminal HA tag:The following specific primers were designed to introduce a Kozakconsensus sequence and a HindIII site immediately upstream of the HA tagand a BamH1 site immediately before the stop codon: Direct primer5′-HindIIIK/HA/fend (SEQ ID NO: 77), and Reverse primer: 3′-fendBamHI(SEQ ID NO: 76). The hydroxylase with a N-terminal HA tag was amplifiedby PCR [5 thermal cycles (1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.)and then 25 thermal cycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68°C.) with KTLA DNA polymerase under standard conditions]. The PCR productwas digested with HindIII and BamH1 and subsequently cloned intoHindIII, BamH1 of pYES2 expression vector.

Lesquerella gracilis bifunctional enzyme: The following specific primerswere designed to introduce a Kozak consensus sequence: Direct primer:HIII NcoI gracilis F (SEQ ID NO: 102), and Reverse primer: gracilis XbaIMfeI R (SEQ ID NO: 101). The hydroxylase was amplified by PCR [5 thermalcycles (1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) and then 25thermal cycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.) with KTLADNA polymerase under standard conditions]. The PCR product was digestedwith HindIII, XbaI and cloned into the HindIII, XbaI of pYES2 yeastexpression vector.

ΔT Lesquerella gracilis bifunctional enzyme: The following specificprimers were designed to introduce a Kozak consensus sequence: Directprimer: HIII NcoI gracilis F (SEQ ID NO: 102), and Reverse primer:gracilis XbaI MfeI R (SEQ ID NO: 101). The ΔT L. gracilis hydroxylasewas amplified by PCR [5 thermal cycles (1 min, 92° C., 30 sec 50° C.,1.5 min 68° C.) and then 25 thermal cycles (1 min, 92° C., 30 sec 57°C., 1.5 min 68° C.) with KTLA DNA polymerase under standard conditions].The PCR product was digested with HindIII, XbaI and cloned into theHindIII, XbaI of pYES2 yeast expression vector.

ΔKKGG Ricinus communis hydroxylase: The following specific primers weredesigned to introduce a Kozak consensus sequence: Direct primer: HIIINcoI castor F (SEQ ID NO: 99), and Reverse primer: castor XbaI MfeI R(SEQ ID NO: 98). The hydroxylase was amplified by PCR [5 thermal cycles(1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) and then 25 thermalcycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.) with KTLA DNApolymerase under standard conditions]. The PCR product was digested withHindIII, XbaI and cloned into the HindIII, XbaI of pYES2 yeastexpression vector.

Crepis biennis epoxygenase enzyme: The following specific primers weredesigned to introduce a Kozak consensus sequence: Direct primer: HIIINcoI C. biennis F (SEQ ID NO: 123), and Reverse primer: Crepis XbaI MfeIR (SEQ ID NO: 103). The hydroxylase was amplified by PCR [5 thermalcycles (1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) and then 25thermal cycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.) with KTLADNA polymerase under standard conditions]. The PCR product was digestedwith HindIII, XbaI and cloned into the HindIII, XbaI of pYES2 yeastexpression vector.

Stokesia laevis epoxygenase enzyme: The following Specific primers weredesigned to introduce a Kozak consensus sequence: Direct primer: BamHINcoI S. epoxygenase F (SEQ ID NO: 106), and Reverse primer: S.epoxygenase XbaI R (SEQ ID NO: 107). The hydroxylase was amplified byPCR [5 thermal cycles (1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) andthen 25 thermal cycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.)with KTLA DNA polymerase under standard conditions]. The PCR product wasdigested with BamHI, XbaI and cloned into the BamHI, XbaI of pYES2 yeastexpression vector.

Nucleotide Sequence Determination

Sequencing of the R. communis hydroxylase, R. communis hydroxylase withN-terminal HA tag, R. communis hydroxylase with C-terminal HA tag, L.lindheimeri bifunctional enzyme, L. lindheimeri bifunctional enzyme withN-terminal HA tag, ΔT L. gracilis, and ΔKKGG R. communis hydroxylasewere performed using an automated sequencer (such as model 373 fromApplied Biosystems, Inc.) using processes well known to those skilled inthe art.

Transformation of Yeast

Transformation was preformed according to the Invitrogen pYES2 kit(V825-20). A fresh yeast culture (initial absorbance=0.4) was grown inYPD medium for 4 hours. The cells were collected and washed once in 1×TE and resuspended in 2 mL of 1× LiAc/0.5×TE (100 mm lithium acetate pH7.5, 5 mm tris-HCL pH 7.5, 0.5 mm EDTA). 100 μg of denatured herringsperm DNA was added as a DNA carrier to 1 μg of the plasmid DNA. 100 μLof competent yeast and 700 μL of 1×liAc/40% PEG-3350/1×TE (100 mMlithium acetate pH 7.5, 40% PEG-3350, 10 mM tris-HCL pH 7.5, 1 mM EDTA)were added. The mixture was incubated at 30° C. for 30 min. 88 μL ofDMSO was added and the mixture was incubated at 42° C. for 7 min. Aftercentrifugation, the cells were resuspended in 1×TE (100 uL) and platedon minimum medium containing suitable supplements.

Over Expression of Genes of Interest in Yeast

Yeast strains transformed with pYES2 plasmid, harboring either no insertor the genes for hydroxylase or bifunctional enzymes were grown at thesame time. For ricinoleic acid analysis, transformed cells were grown inSC-URA (yeast synthetic complete media devoid uracil, Sigma)supplemented with 2% glucose and 1% casamino acids at 30° C. to anoptical density (600 nm) of 2.5. Cells were then centrifuged, washed 3times in SC-URA media containing no glucose and cultured for 48 hours at30° C. on SC-URA media (yeast synthetic complete media devoid of uracil,Sigma) supplemented with 2% galactose and 1% casamino acids. Cultureswere centrifuged and dried.

Fatty Acid Analysis of Yeast Extracts

Dried yeast pellets were methylated with (400 μL1% sodium methoxide inmethanol), extracted with hexane, and trimethylsilylated (100 μLBSTAFA-TMCS, Supelco, 90° C. for 45 minutes). Samples were analyzed onan Agilent 6890 GC-5973 Mass Selective Detector (GC/MS) and an AgilentDB-23 capillary column (0.25 mm×30 m×0.25 um). The injector was held at250° C., the oven temperature was 235° C., and a helium flow of 1.0mL/min was maintained.

Table 9 shows examples of MS data from yeast expressing some of theenzymes described in Example 13.

TABLE 9 Ricinus communis hydroxylase with or without an N-terminal HAtag: Construct % R 3522 6.7 3522 4.1 3522 4.8 3522 9.5 3522 4.6 4074*2.1 4074* 3.0 4074* 5.3 4074* 3.2 *Designates a construct carrying anN-terminal HA tag.

These GC/MS data indicate that the hydroxylase from R. communis (3522 or4074*) was functional when expressed in yeast. The percentages ofricinoleic acid (% R) listed in the table are percentages of the totalfatty acid.

TABLE 10 L. gracilis bifunctional hydroxylase expressed in yeastConstruct % R 3958 8.0 3958 8.2 3958 13.1 3958 12.2 3958 10.7 3958 9.23958 6.3

These GC/MS data indicate that the hydroxylase from L. gracilus (3958)was functional when expressed in yeast. The percentages of ricinoleicacid (% R) listed in the table are percentages of the total fatty acid.

TABLE 11 ΔT Lesquerella gracilis bifunctional hydroxylase expressed inyeast Construct % R 4323 5.9 4323 5.9 4323 8.2 4323 7.2 4323 7.4

These GC/MS data indicate that the hydroxylase from L. gracilis wasfunctional when expressed in yeast despite the deletion of amino acid83. The percentages of ricinoleic acid (% R) listed in the table arepercentages of the total fatty acid.

TABLE 12 ΔKKGG castor hydroxylase expressed in yeast Construct % R 43030.7 4303 1.4 4303 1.5 4303 1.3 4303 1.5

These GC/MS data indicate that the deletion mutant hydroxylase (ΔKKGG)from R. communis was functional when expressed in yeast despite theamino acid deletions at positions 18-21. The percentages of ricinoleicacid (% R) listed in the table are a percentage of the total fatty acid.

TABLE 13 Negative Control Construct % R % O 3677 0 35.8 3677 0 34.713677 0 36.34 3677 0 30.87 3677 0 30.16

These GC/MS data indicate that no detectable amounts of ricinoleic acidwere produced when the vector with no insert was expressed in yeast. Thepercentages of ricinoleic (% R) and oleic acid (% O) listed in the tableare percentage of the total fatty acid.

EXAMPLE 16

This example describes the construction of vectors suitable forexpression in plants. Schematic diagrams of the vectors are shown inFIGS. 4-6.

Generation of Transgenic Vectors: Building Modified pUCAP Vectors

The pUCAP vector [Engelen et al. (1995) Transgenic Res. 4(4):288-290]was modified to create pUCAP2, pUCAP3, pUCAP4, pUCAP5, and pUCAP6.

The following specific primers were designed to introduce a 5′-SacI anda 3′-EcoRI site flanking the Ubi3 terminator: Direct primer: UT3 (SEQ IDNO: 78), and Reverse primer: UT4 (SEQ ID NO: 79). The Ubi3 terminatorwas amplified from pBinplus [Engelen et al. (1995) Transgenic Res.4(4):288-290] by PCR [25 cycles (4 min 94° C., 30 sec 60° C., 1 min 68°C.) with KTLA DNA polymerase under standard conditions]. The PCR productwas digested with SacI and EcoRI and subsequently cloned into pUCAP togive pUCAP1.

The following specific primers were designed to introduce a 5′-AscI anda 3′-SphI site flanking the Ubi3 promoter: Direct primer: UP1 (SEQ IDNO: 80), and Reverse primer: UP2 (SEQ ID NO: 81). The Ubi3 promoter wasamplified from pBinplus [Engelen et al. (1995) Transgenic Res.4(4):288-290] by PCR [25 cycles (4 min 94° C., 30 sec 60° C., 1 min 68°C.) with KTLA DNA polymerase under standard conditions]. The PCR productwas digested with AscI and SphI and subsequently cloned into theAscI/SphI sites of pUCAP1 giving pUCAP2.

The following specific oligos were designed to create an HA tag with aBamH1 overhang immediately before the initiation codon and a SacIoverhang immediately after the last codon of the tag: Direct oligo: CHA1(SEQ ID NO: 84), and Reverse oligo: CHA2 (SEQ ID NO: 85). The HA tag wascreated by annealing oligos (0.1 pg/uL) at 92° C. for 3 minutes andslowly bringing to room temperature. The HA tag was cloned into theBamHI/SacI sites of pUCAP2 to create pUCAP3. DNA cloned into the MCS ofpUCAP3 will have the HA tag at the C-terminus.

The following specific oligos were designed to create an HA tag with aHindIII overhang immediately before the initiation codon and a SalIoverhang immediately after the last codon of the tag: Direct oligo: HA5(SEQ ID NO: 82), and Reverse oligo: HA6 (SEQ ID NO: 83). The HA tag wascreated by annealing oligos (0.1 pg/uL) at 92° C. for 3 minutes andslowly bringing to room temperature. The HA tag was cloned into theHindIII/SalI site of pUCAP2 to create pUCAP4. DNA cloned into the MCS ofpUCAP4 will have the HA tag at the N-terminus.

The following specific primers were designed to add a 5′-AscI site and a3′-SphI site flanking the A. thaliana IRT2 promoter to AscI and SphI ofpUCAP1: Direct primer: IRT 1 (SEQ ID NO: 86), and Reverse primer: IRT2(SEQ ID NO: 87). The IRT2 promoter was amplified from Arabidopsisthaliana using a 30 cycle Gradient PCR [(4 min 95° C., 30 sec 48-63° C.,2 min 68° C.) with KTLA DNA polymerase under standard conditions]. ThePCR product was digested with AscI/SphI and AscI/SphI cloned into ofpUCAP1 giving pUCAP5.

pUCAP6 was created by replacing the Ubi3 promoter of pUCAP3 with theIRT2 promoter, using the AscI/SphI sites.

Generation of a Vector Containing the HA-Tag for N-Terminal Fusions.

The_oligonucleotides HA-tag-F (SEQ ID NO: 109) and HA-tag-R (SEQ ID NO:110) were mixed and annealed using standard procedures. The annealedproduct generates compatible ends for HindIII and BamHI restrictionsites and was cloned into the plasmid vector pUC118, generating theplasmid pUC-HA.

Plant Transformation Vector Containing the 5′ UTR and 3′ UTR Regions ofthe fad2 Gene from A. thaliana.

A. thaliana genomic DNA was used as template and KTLA was the DNApolymerase of choice For PCR. Primers Fad5′UTR-F (SEQ ID NO: 111) andFad5′UTR-R (SEQ ID NO: 112) were used to PCR amplify the 5′ UTR, firstintron and first codon of fad2, flanked by the restriction sites XhoI atthe 5′ end and NcoI, BamHI at the 3′ end. PCR reactions were performedunder standard conditions as follow: 97° C. for 30 sec, 35 cycles ofamplification (45 sec at 94° C., 1 min at 55° C., 90 sec at 72° C.) anda final extension of 5 min at 72° C. The PCR product was cloned into theplasmid vector pCR2.1 (Invitrogen).

Primers Fad3′UTR-F (SEQ ID NO: 113) and Fad3′UTR-R (SEQ ID NO: 114) wereused to PCR amplify the 3′ UTR of fad2. Reactions were performed asfollow: 97 C for 10 sec and 35 cycles of amplification (30 sec at 94°C., 1 min at 60° C., 2.5 min at 72° C.). The PCR product was cloned intothe plasmid vector pCR2.1. The identities of both PCR products, fad2 5′UTR (SEQ ID NO: 44) and Fad2 3′ UTR (SEQ ID NO: 45) were confirmed byDNA sequencing.

The plant transformation vector containing both fad2 UTR regions wasconstructed in two steps: first, the fad2 5′ UTR fragment was subclonedimmediately downstream of the CaMV35S promoter of a binary vector as aXhoI/BamHI insert. Then, the A. tumefaciens NOS 3′ UTR present in theplasmid between the XbaI and HindIII restriction sites was replaced withthe A. thaliana fad2 3′UTR fragment, between the same sites, generatinga plasmid called pFADUTR.

Cloning Hydroxylase and Bifunctional Hydroxylase Genes into pUCAP3pUCAP4 and pUCAP6

R. communis hydroxylase and L. lindheimeri bifunctional hydroxylasegenomic clones were generated by PCR amplification using specificprimers.

Ricinus communis hydroxylase with a C-terminal HA tag: The followingspecific primers were designed to introduce a HindIII site immediatelyupstream of the initiation codon and a BamHI site immediately beforestop codon: Direct primer: Castor 5′-HindIII (SEQ ID NO: 88), andReverse primer: Castor 3′-BamHI (SEQ ID NO: 89). The hydroxylase wasamplified by PCR [5 cycles (4 min 94° C., 45 sec 94° C., 50° C. 45 sec,72° C.) and then 25 cycles (45 sec 94° C., 45 sec 58° C., 2 min 72° C.)with KTLA under standard conditions]. The PCR product was digested withHindIII and BamH1 and subsequently cloned into HindIII, BamH1 of pUCAP3expression vector giving Rc-pUCAP3.

Ricinus communis hydroxylase with a N-terminal HA tag: The followingprimers were designed in order to tag the Ricinus communis hydroxylasewith a N-terminal HA tag: Direct primer: BamHI castor F (SEQ ID NO:104), and Reverse primer: castor XbaI MfeI R (SEQ ID NO: 98). Thehydroxylase gene was amplified by PCR [5 thermal cycles (1 min, 92° C.,30 sec 50° C., 1.5 min 68° C.) and then 25 thermal cycles (1 min, 92°C., 30 sec 57° C., 1.5 min 68° C.) with KTLA DNA polymerase understandard conditions]. The PCR product was digested with BamHI/MfeI andsubcloned into the BamHI/EcoRI sites of the pUC-HA vector.

Lesquerella lindheimeri bifunctional enzyme with an N-terminal HA tag:The following specific primers were designed to introduce a SalI siteimmediately upstream of the start codon and BamH1 site immediately afterthe stop codon: Direct primer fend F SalI (SEQ ID NO: 90), and Reverseprimer: Fend R B-stop. (SEQ ID NO: 91). The bi-functional hydroxylasegene was amplified by PCR [5 cycles (4 min 94° C., 45 sec 94° C., 45 sec50° C., 2 min 72° C.) and then 25 cycles (45 sec 94° C., 45 sec 58° C.,2 min 72° C.) with KTLA DNA polymerase under standard conditions]. ThePCR product was digested with SalI and BamH1 subsequently cloned intoSail/BamH1 of pUCAP4 giving Rc-pUCAP4.

L. gracilis bifunctional hydroxylase with a N-terminal HA tag: Thefollowing primers were designed in order to tag the L. gracilisbifunctional hydroxylase with a N-terminal HA tag, Direct primer: BamHIgracilis F (SEQ ID NO: 105), and Reverse primer: gracilis XbaI MfeI R(SEQ ID NO: 101). The hydroxylase gene was amplified by PCR [5 thermalcycles (1 min, 92° C., 30 sec 50° C., 1.5 min 68° C.) and then 25thermal cycles (1 min, 92° C., 30 sec 57° C., 1.5 min 68° C.) with KTLADNA polymerase under standard conditions]. The PCR product was digestedwith BamHI/MfeI and subcloned into the BamHI/EcoRI sites of the pUC-HAvector.

The Crepis biennis and Stokesia laevis epoxygenase genes were subclonedas described above into the pUC-HA vector using BglII NcoI C. biennis F(SEQ ID NO: 108)/Crepis XbaI MfeI R (SEQ ID NO: 103) and BamHI NcoI S.epoxygenase F (SEQ ID NO: 106)/S.epoxygenase XbaI R (SEQ ID NO:107).

The Crepis biennis and Stokesia laevis epoxygenase genes lacking the HAsequence were subcloned as described above into a plant expressionvector using AscI NcoI C. biennis F (SEQ ID NO: 124)/Crepis XbaI MfeI R(SEQ ID NO: 103) and BamHI NcoI S. epoxygenase F (SEQ ID NO: 106)/S.epoxygenase XbaI R (SEQ ID NO:107).

Plant Expression Vectors

Constructs Rc-pUCAP3, Ll-pUCAP4, and Rc-pUCAP6 were digested with AscIand PacI to release the inserts and inserts were subsequently sub-clonedinto the AscI/PacI sites of pBinPlusARS binary vector engineered asdescribed by [Engelen et al. (1995) Transgenic Res. 4(4):288-290] givingRc-3pBinPlusARS, L/4-pBinPlusARS and Rc6-pBinPlusARS.

ΔKKGG castor, ΔT gracilis, R. communis hydroxylase, chimeric fad2/R.communis hydroxylase, L. gracilis bifunctional hydroxylase, chimericfad2/L. gracilis bifunctional hydroxylase, C. biennis epoxygenase, andS. laevis epoxygenase genes were subcloned into a plant expressionvector using NcoI/XbaI restriction enzyme sites. N-terminal HA taggedchimeric fad2/R. communis hydroxylase and N-terminal chimeric fad2/R.communis hydroxylase were removed from pUC-HA and subcloned into a plantexpression vector using NcoI/XbaI restriction enzyme sites. The aboveconstructs were also subcloned into a plant expression vector containingthe fad2 5′UTR and fad2 3′UTR (pFADUTR), using the NcoI/XbaI restrictionsites.

EXAMPLE 17

This example describes the production of transgenic Arabidopsis plants,transgenic tomato callus, transgenic tomato hairy roots, Arabidopsishairy root, soybean hairy root, and soybean composite plants using theplasmid vectors described in Example 16.

Transformation of Agrobacterium tumefaciens and Agrobacterium rhizogenes

Plant expression vectors harboring genes encoding hydroxylases,epoxygenases or chimeric fad2 constructs were transformed intoAgrobacterium tumefaciens LB4404 as follows. Agrobacterium was grownovernight in 100 mL of LB [(1% bacto tryptone, 0.5% sodium chloride and0.5% bacto-yeast extract) supplemented with kanamycin (50 ug/mL),rifampicin (10 ug/mL), and streptomycin (150 ug/mL)]. 100 mL of LBsupplemented in the same manner was inoculated with 1 mL of theovernight culture and grown at 30° C. for 4 hrs. The culture was chilledfor 10 minutes and cells were harvested by centrifugation. Cells wereresuspended in 1 mL of ice cold CaCl₂ (20 mM) and dispensed into 100 μLaliquots. 1 μg of plasmid DNA was added to the cells, frozen on dry ice,put at 37° C. for 5 minutes, and shaken for 90 minutes at 30° C. in 1 mLLB. Cells were pelleted and resuspended in 100 μL of LB and plated on LBplates [(1% bacto tryptone, 0.5% sodium chloride, 0.5% bacto-yeastextract, and 0.15% agar) supplemented with kanamycin (50 ug/mL),rifampicin (10 ug/mL), and streptomycin (150 ug/mL)].

Transformation of Agrobacterium rhizogenes strain A4 was performed inthe same manner as Agrobacterium tumefaciens strain LB4404 with thefollowing exceptions: Media used was MGL [extract (2.5 g/L), tryptone (5g/L), sodium chloride (5 g/L), L-glutamic acid (1 g/L), mannitol (5g/L), potassium phosphate (0.26 g/L), magnesium sulfate heptahydrate(100 mg/L), and biotin (1 mg/L)] and MGL plates [yeast extract (2.5g/L), tryptone (5 g/L), sodium chloride (5 g/L), L-glutamic acid (1g/L), mannitol (5 g/L), potassium phosphate (0.26 g/L), magnesiumsulfate heptahydrate (100 mg/L), biotin (1 mg/L), and bacto-agar (14g/L)].

Plant Transformation

Arabidopsis thaliana was transformed via Agrobacterium tumefaciensfollowing Clough and Bent [Clough & Bent (1998) Plant J. 19(3):249-257].Briefly, 5 mL overnight cultures of transformed LB4404 (LB-10 ug/mLrifampicin, 50 ug/mL kanamycin, 150 μg/mL streptomycin) were grown at30° C. The 5 mL cultures were used to inoculate 500 mL LB (10 μg/mLrifampicin, 50 μg/mL kanamycin, 150 ug/mL streptomycin) and grownovernight at 30° C. Cultures were spun down (5K, 5 min). Pellets wereresuspended in 5% glucose+0.02% Silwet L-77. The above ground parts ofthe plant were submerged into Agrobacterium solution for 5 min withgentle agitation. Plants were covered under a dome overnight.

Fatty Acid Analysis of Arabidopsis thaliana Leaf and Root Tissue

Generation of Plant Material

Seed sterilization: Approximately 200 second generation seeds fromtransformed plants were placed in an eppendorf tube. 1 mL of 20% bleachin ethanol was added and the tubes were left at room temperature for 15minutes. The seeds were then washed 2× with 100% ethanol and openedtubes were left in the laminar flow hood to dry overnight.

Seed Germination: Approximately 50 seeds were placed on 0.5×MS plates,wrapped in parafilm, and kept at room temperature until germination.

Approximately 0.10 g of root tissue or leaf tissue was put in a 1.5 mLeppendorf tube and frozen on dry ice and subsequently ground with apestle. The ground root tissue was then methylated with (500 μL 1%sodium methoxide in methanol), extracted with hexane, andtrimethylsilylated (100 μL BSTAFA-TMCS, Supelco, 90° C. for 45 minutes).Samples were analyzed on an Agilent 6890 GC-5973 Mass Selective Detector(GC/MS) and an Agilent DB-23 capillary column (0.25 mm×30 m×0.25 um).The injector was held at 250° C., the oven temperature was 235° C., anda helium flow of 1.0 mL/min was maintained.

TABLE 14 Fatty Acid Analysis of extracts from Arabidopsis thalianaharboring a chimeric fad2/R. communis hydroxylase Tissue Construct Line% R % L % O Leaves 4028* 6 1.19 15.82 1.87 Leaves 4028* 6 1.11 15.222.02 Roots 4028* 6 0.54 25.52 0.61 Roots 4028* 6 0.10 22.24 0.73 Roots4062  3 1.44 21.18 5.09 Roots 3819  — 0 21.54 1.96 *Designatesconstructs with a HA tag on the N-terminus.

These GC/MS data indicate that a chimeric fad2/R. communis hydroxylase(4062 or 4028*) operably linked to 5′ and 3′fad2 UTRs was functionalwhen expressed in A. thaliana. The percentages of ricinoleic acid listedin the table are a percentage of the total fatty acid. A. thalianatransformed with a vector containing no insert (3819), did notaccumulate ricinoleic acid (R).

Hairy Root Transformation Protocol for Tomato

Plant material preparation: This protocol can be used for tomato roottransformation. Numerous strains of A. rhizogenes may be used as thetransforming agent, however, strain A4 (ATCC number 43057) was used inthis case. Lycopersicon esculentum cv. Rutgers, Money Maker or MountainSpring, were used, although other varieties that are susceptible toMeloidogyne incognita (M. incognita) infection may be used. As acontrol, the resistant cultivar Motelle was used [Vos et al. (1998) Nat.Biotechnol. 16: 1365-1369]. This protocol can also be used to generatehairy root cultures from Arabidopsis thaliana, ecotype Columbia.

The transformation protocol is similar to that described previously[McCormick (1991) Transformation of tomato with Agrobacteriumtumefaciens. in Plant Tissue Culture Manual, Fundamentals andApplications, K. Lindsey (ed), Kluwer, Vol. B6: 1-9]. Briefly, tomatoseeds were sterilized with hypochlorite and grown in magenta boxescontaining Gamborg's synthetic medium [Gamborg et al. (1968) Exp. CellRes. 50:151-158] in daylight for 7 days, until cotyledons are completelyunfolded. Cotyledons were removed sterilely and wounded in MSO medium(MS salts, 3% sucrose, Gamborg's B5 vitamins, pH 5.8) by removing boththe proximal and distal tips with a razor blade. Wounded cotyledons wereincubated for 1-2 days, adaxial side up, on filter paper placed on 150mm² plates made with D1 medium (MS salts, 3% glucose, Gamborg's B5vitamins, 1 mg/L zeatin, 0.8% Gel-rite agar). After this incubationperiod, cotyledons were cocultured with a suspension of A. rhizogenes toinitiate transformation.

A. rhizogenes culture preparation: A glycerol stock of A. rhizogenes A4was streaked onto MGL medium [McCormick (1991) Transformation of tomatowith Agrobacterium tumefaciens. in Plant Tissue Culture Manual,Fundamentals and Applications, K. Lindsey (ed), Kluwer, Volume B6: 1-9]and grown at 29° C. until individual colonies appeared. A single colonywas used to inoculate a 15 mL culture of MGL medium, which was grown forone day in a shaking incubator at 29° C., 100 rpm. On the following day,the bacteria were harvested by centrifugation at 3800×g for 10 minutes.The resulting pellet was washed twice, without disturbing the pellet,with 15 mL of MSO medium and centrifuging at 3800×g for 5 minutes. Thefinal pellet was resuspended in 15 mL MSO medium and the optical densityof the culture at 550 nm was determined. The density was adjusted to 0.4with MSO medium. 10 mL of this culture was used for cocultivation afterthe addition of 50 μl of 0.074 M acetosyringone. Cocultivation wasperformed within one hour of the addition of acetosyringone.

Cocultivation of tomato cotyledons and A. rhizogenes: Onto each plate ofcotyledons, 5 mL of A. rhizogenes culture was pipetted over thepreincubated cotyledons using sterile technique. The plates wereincubated at room temperature for 10 minutes, with occasional swirlingof plates during this time. The bacterial suspension was then removedwith a sterile pipette. The cotyledons were transferred gently, abaxialside up, using a scalpel or razor blade, to a new 100×20 mm Petri platecontaining a Whatman filter paper disk on D1 medium. The plates weresealed with micropore tape and incubated for 2 days at room temperaturenear a south facing window.

Selection of transgenic roots: After cocultivation, the cotyledons weretransferred, abaxial side up onto Gamborg's medium containing 200 mg/Lcefotaxime at a density of 20-30 cotyledons per plate. The plates weresealed with micropore tape and incubated at room temperature in the darkfor 10 days. On the 10^(th) day, the cotyledons were transferred tofresh selective media plate. After an additional 10 day period, hairyroot initials were removed from the cotyledons using a sterile razorblade and incubated on selective medium with transfer to fresh platesafter 10 days. To assess whether the hairy roots were cured of infectionby A. rhizogenes, the roots were transferred to Gamborg's medium withoutcefotaxime and allowed to grow for 10 days. Any plates showing bacterialgrowth around the roots were discarded.

Root cultures were maintained on Gamborg's medium lacking selection byserial transfer every 20-30 days.

Fatty Acid Analysis of Tomato Hairy Root Extracts

Approximately 0.25 g of root tissue was placed in a 1.5 mL eppendorftube and frozen on dry ice and subsequently ground with a pestle. Theground root tissue was then methylated with (500 μL1% sodium methoxidein methanol), extracted with hexane, and trimethylsilylated (100 μLBSTAFA-TMCS, Supelco, 90° C. for 45 minutes). Samples were analyzed onan Agilent 6890 GC-5973 Mass Selective Detector (GC/MS) and an AgilentDB-23 capillary column (0.25 mm×30 m×0.25 um). The injector was held at250° C., the oven temperature was 235° C., and a helium flow of 1.0mL/min was maintained.

TABLE 15 Fatty Acid Analysis of tomato roots harboring a R. communishydroxylase Construct Line % R % L % O Temp Cultivar 4203 7 1.637 50.540.94 23 Money Maker 4203 7 1.17 50.48 1.20 23 Money Maker 4203 16 1.2955.67 0.00 23 Money Maker 4203 16 1.07 52.04 1.89 23 Money Maker 4203 151.21 53.66 1.25 23 Money Maker 4203 15 0.91 51.57 1.63 23 Money Maker3677 19 0 47.06 0.00 23 Money Maker

These GC/MS data indicate that a R. communis (4203) hydroxylase wasfunctional when expressed in tomato hairy root tissue. The percentagesof ricinoleic acid (% R) listed in the table are percentages of thetotal fatty acid. Tomato hairy roots transformed with a vectorcontaining no insert (3677), did not accumulate ricinoleic acid (R).Linoleic and oleic acid percentages are listed under the columns % L and% O, respectively.

TABLE 16 Fatty Acid Analysis of tomato roots harboring a chimericfad2/R. communis hydroxylase Construct Line % R % L % O Temp Cultivar3927 7 2.81 49.02 2.05 23 Rutgers 3927 7 1.97 51.78 2.22 23 Rutgers 39277 1.67 55 2.17 23 Rutgers 3927 20 1.03 52.38 1.04 15 Rutgers 3927 200.98 51.08 1.59 15 Rutgers 3927 20 0.75 50.89 1.14 23 Rutgers  3938* 141.02 47.92 1.25 23 Rutgers  3938* 14 0.973 48.57 2.25 23 Rutgers  3938*18 0.49 49.45 1.45 23 Rutgers  3938* 18 0.86 47.98 2.16 23 Rutgers 36770 52.05 2.51 23 Rutgers *Designates HA on N terminus

These GC/MS data indicate that a chimeric fad2/R. communis hydroxylase(3927 or 3938*) was functional when expressed in tomato hairy root. Thepercentages of ricinoleic acid (% R) listed in the table are percentagesof the total fatty acid. Tomato hairy roots transformed with a vectorcontaining no insert (3677) did not accumulate ricinoleic acid (R).Linoleic and oleic acid percentages are listed under the columns % L and% O, respectively.

TABLE 17 Chimeric fad2/R. communis hydroxylase with 5′ and 3′ fad2 UTRsConstruct Line % R % L % O Temp Cultivar 4062 19 1.26 48.04 6.99 23Rutgers 4062 19 2.25 48.22 4.59 23 Rutgers 4062 19 1.97 50.19 3.60 23Rutgers  4028* 12 2.38 50.54 2.43 15 Rutgers  4028* 12 2.36 52.64 2.7015 Rutgers  4028* 12 1.13 51.34 4.19 23 Rutgers 3677 2 0 53.32 0.84 RTRutgers  4028* 5 0.95 53.15 2.49 RT Mountain Spring  4028* 5 1.3 54.81.55 RT Mountain Spring  4028* 5 0.58 47.61 2.56 RT Mountain Spring 36772 0 57.94 0.87 RT Mountain Spring *Designates HA on N terminus. RT =room temperature

These GC/MS data indicate that a chimeric Fad2/R. communis hydroxylase(4062 or 4028*) operably linked to 5′ and 3′ fad2 UTRs was functionalwhen expressed in tomato hairy root. The percentages of ricinoleic acidlisted in the table are percentages of the total fatty acid. Tomatohairy roots transformed with a vector containing no insert (3677) didnot accumulate ricinoleic acid (R).

Hairy Root Transformation Protocol for soybean

Seed sterilization: Approximately 250 seeds were placed in a 100×25 mmplate and placed in a desicator in a fume hood. Using a 350 mL beaker, 2mL of concentrated HCl was carefully added to 200 mL of 100% bleach andthe beaker was placed inside the desicator to expose the seeds tosterilizing gas. After 24 hours, the procedure was repeated. This wasdone 3 times for a total of 3 sterilizations. To test for sterility, 10seeds were placed in LB and put in a shaker at 37° C. for 24 hour. Ifthe LB was clear, indicating no bacterial growth, the seeds were sealedin the Petri dish and germinated at a later date. If there was bacterialgrowth, the sterilization procedure was performed again.

Seed Germination: 9 seeds were placed on 0.25× solid MS plates, wrappedin parafilm, and kept at room temperature for 7 days.

A. rhizogenes culture preparation: A glycerol stock of A. rhizogenes A4was streaked onto MGL medium [McCormick (1991) Transformation of tomatowith Agrobacterium tumefaciens. in Plant Tissue Culture Manual,Fundamentals and Applications, K. Lindsey (ed), Kluwer, Volume B6: 1-9]and grown at 29° C. until individual colonies appeared. A single colonywas used to inoculate a 15 mL culture of LB+Kanamycin medium, which wasgrown for one day in a shaking incubator at 29° C., 100 rpm. On thefollowing day, the bacteria were harvested by centrifugation at 3800×gfor 10 minutes. The resulting pellet was resuspended in MSO to a finaloptical density of 0.2-0.3. Acetosyringone was then added to a finalconcentration of 375 um. Cocultivation was performed within one hour ofthe addition of acetosyringone.

Explant Excision: The cotyledons were cut from the main axis making surethat the axillary bud was removed.

Cocultivation of soybean cotyledons and A. rhizogenes: Soybeancotyledons were added to the culture using sterile technique. Thecultures were then vacuum infiltrated for 2 minutes and incubated atroom temperature for 20 minutes. The bacterial suspension was thenremoved with a sterile pipette. The cotyledons were transferred gently,abaxial side up, using tweezers, to a 100×20 mm Petri plate containing aWhatman filter paper disk soaked in MSO. The plates were sealed withmicropore tape and incubated for 2 days at room temperature near a southfacing window.

Selection of transgenic roots: After cocultivation, the cotyledons weretransferred, abaxial side up onto MS solid medium containing 500 mg/Lcarbenicillin at a density of 10 cotyledons per plate. The plates weresealed with micropore tape and incubated at room temperature. About 28days post-inoculation, hairy roots were removed from the cotyledonsusing a sterile razor blade and incubated on Gamborgs medium plusselection.

Hairy Root Transformation Protocol for Arabidopsis thaliana

Seed sterilization: Approximately 200 seeds were placed in an eppendorftube. 1 mL of 20% bleach in ethanol was added and the tubes were left atroom temperature for 15 minutes. The seeds were then washed 2× with 100%ethanol and opened tubes were left in the laminar flow hood to dryovernight.

Seed Germination: Approximately 50 seeds were placed on 0.5× solid MSplates, wrapped in parafilm, and kept at room temperature untilgermination.

A. rhizogenes culture preparation: A glycerol stock of A. rhizogenes A4was streaked onto MGL medium [McCormick (1991) Transformation of tomatowith Agrobacterium tumefaciens. in Plant Tissue Culture Manual,Fundamentals and Applications, K. Lindsey (ed), Kluwer, Volume B6: 1-9]and grown at 29° C. until individual colonies appeared. A single colonywas used to inoculate a 15 mL culture of LB+Kanamycin medium, which wasgrown for one day in a shaking incubator at 29° C., 100 rpm. On thefollowing day, the bacteria were harvested by centrifugation at 3800×gfor 10 minutes. The resulting pellet was resuspended in MSO to a finaloptical density of 0.2-0.3. Acetosyringone was then added to a finalconcentration of 375 um. Cocultivation was performed within one hour ofthe addition of acetosyringone.

Explant Excision: A. thaliana cotyledons were removed sterilely andwounded in MSO medium (MS salts, 3% sucrose, Gamborg's B5 vitamins, pH5.8) by removing both the proximal and distal tips with a razor blade.Wounded cotyledons were incubated for 1-2 days, adaxial side up, onfilter paper placed on 150 mm² plates made with D1 medium (MS salts, 3%glucose, Gamborg's B5 vitamins, 1 mg/L zeatin, 0.8% Gel-rite agar).After this incubation period, cotyledons were cocultured with asuspension of A. rhizogenes to initiate transformation.

Cocultivation of A. thaliana cotyledons and A. rhizogenes: A. thalianacotyledons were added to the A. rhizogenes culture using steriletechnique and left at room temperature for 10 minutes. The bacterialsuspension was then removed with a sterile pipette. The cotyledons weretransferred gently, abaxial side up, using a sterile spatula, to aWhatman filter paper disk in a 100×20 mm Petri plate containing solidGamborgs medium plus 500 mg/L carbenicillin. The plates were sealed withmicropore tape and incubated for at room temperature near a south facingwindow.

Selection of transgenic roots: About 10 days post-inoculation, hairyroots were removed from the cotyledons using a sterile razor blade andplaced on Gamborgs medium plus selection.

Callus Transformation Protocol

Plant material preparation: This protocol can be used to generatetransgenic tomato callus. All transformations carried out usedAgrobacterium tumefaciens strain LB4404 and the tomato cultivarLycopersicon esculentum cv. Rutgers, Money Maker, or Mountain Spring.Tomato cotyledons were grown as described in the hairy roottransformation section.

A. tumefaciens culture preparation: A glycerol stock of A. tumefaciensLB4404 was streaked onto LB medium (rifampicin 10 mg/L, streptomycin 150mg/L, kanamycin 50 mg/L) (McCormick, 1991) and grown at 29° C. untilindividual colonies appeared. A single colony was used to inoculate a 15mL culture of LB medium, which was grown for one day in a shakingincubator at 29° C., 100 rpm. On the following day, the bacteria wereharvested by centrifugation at 3800×g for 10 minutes. The resultingpellet was washed twice, without disturbing the pellet, with 15 mL ofMSO medium and centrifuging at 3800×g for 5 minutes. The final pelletwas resuspended in 15 mL MSO medium and the optical density of theculture at 550 nm was determined. The density was adjusted to 0.4 withMSO medium. 10 mL of this culture was used for cocultivation after theaddition of 50 μL of 0.074 M acetosyringone. Cocultivation was performedwithin one hour of the addition of acetosyringone.

Cocultivation of tomato cotyledons and A. tumefaciens: Cocultivation wascarried out as described in the hairy root transformation section withthe exception of using A. tumefaciens.

Selection of transgenic callus: After cocultivation, the cotyledons weretransferred, abaxial side up onto 2Z medium (4.3 g MS salt/L, 20%sucrose, 1 mg zeatin/L, 100 mg/L inositol, 1× Nitsch vitamin, 1× folicacid, 8 g/L tissue culture agar) containing 200 mg/L cefotaxime and 100mg/L kanamycin at a density of 20-30 cotyledons per plate. The plateswere sealed with micropore tape and incubated at room temperature in thedark for 10 days. Every 10 days, the cotyledons were transferred tofresh selective media plate. Explants started to grow green or whitecallus after two to three weeks. Explants that were dying (turningbrown) were removed. Callus was excised from explants that containeddying tissue. The callus was maintained on Gamborg's medium.

Composite Plant Protocol for Soybean:

Agrobacterium rhizogenes A4 cultures were grown overnight at 30° C. inLuria Broth with the appropriate antibiotics. Cultures were spun down at4,000 g for 10 minutes. Cells were suspended with ¼ MS to a finalO.D._(600nm) between 0.2-0.5.

Sterile soybean seeds (Cl₂ gas treated seeds) were planted in soil.Young shoots lacking any inflorescences were cut in the middle of theinternode region. Shoots were transplanted into one cm² FibrGro® cubes.Each transplant was inoculated with 4 mL of suspended A. rhizogenes,placed in a flat, covered with a clear lid, and left on the bench topfor one day to allow for acclimation. On the second day the lid wasremoved to let the cubes dry out. Transplants were then watered andcovered. Roots appeared between two and four weeks. Transformed rootscan be identified by a visible marker. The untransformed roots should beexcised. After several weeks, shoots can be transplanted to sand fornematode infection assays.

EXAMPLE 18

This example describes assays to measure anthelmintic activity oftransgenic plants.

Infection of hairy roots: Plates for assays were prepared bytransferring one growing hairy root tip, 1-2 cm long, from a stock rootplate onto 100×15 cm Petri dishes containing approximately 30 mL ofGamborg's media in which the Gel-rite agar had been replaced by 3.0%Phytagel (Sigma catalog P-8169). At least two plates were used pertransgenic line per assay. As a control, we used a hairy root line thatwas generated using A. rhizogenes that had been transformed with a planttransformation plasmid that does not carry any coding sequence after thepromoter. Assay plates were sealed with micropore tape and incubated at28° C. for 4-7 days prior to infection with Meloidogyne incognita eggs.

Preparation of Meloidogyne incognita inoculum: M. incognita eggs wereharvested from a greenhouse-grown tomato plant (Lycopersicon esculentumcv. Mountain Spring) that had been infected 28-42 days previously with5000 M. incognita eggs using a protocol described previously [Hussey &Barker (1973) Plant Disease Reporter 57:1025-1028]. Aerial tissues ofthe tomato plant were removed and the root mass was freed from soil bygentle agitation in a bucket filled with tap water. The root mass wastransferred to a household blender with the addition of 500 mL 10%bleach solution (Clorox bleach in tap water) and chopped into finepieces using the puree setting. The root slurry was transferred to a 200mesh sieve seated on top of a 500 mesh sieve (VWR catalog numbers57334-480 and 57334-492, respectively) and eggs were collected on the500 mesh sieve by rinsing vigorously with tap water. Eggs were furthercleaned and concentrated by sucrose density centrifugation. Eggs werecollected in approximately 30 mL of water and were pipetted on top of 30mL of 30% sucrose solution in a 50 mL centrifuge tube and banded bycentrifugation in a swinging bucket rotor at 1000×g for 10 minutes. Theeggs were collected using a Pasteur pipette and rinsed extensively toremove sucrose on a small 500 mesh sieve using tap water. Eggs werecollected in a small amount of water and stored at 4° C. until use.

Sterilization of inoculum: Approximately 100,000 stored M. incognitaeggs were placed in a 15 mL centrifuge tube and brought to 10 mL volumewith a 10% bleach solution. The tube was agitated for 5 minutes and eggswere collected by centrifugation as described above. The supernatant wasremoved and the eggs were rinsed 3 times with sterile water. Eggs wereresuspended in 1 mL of water and counted using a McMaster worm eggcounting chamber. Only eggs containing vermiform larvae were counted.

Alternatively, if hatched J2 larvae were to be used as inoculum, eggswere hatched using a standard protocol. Larvae were collected bycentrifugation as above and sterilized as described in Atkins, 1996[Atkinson et al. (1996) J. Nematol. 28:209-215], using sequentialincubations in penicillin, streptomycin sulfate, and chlorhexidinesolutions, followed by rinsing in sterile water.

Inoculation and monitoring of assay: Hairy root infections wereinitiated by adding either 300 eggs or 100 J2 larvae per plate in 10 μL,using sterile technique. Plates were resealed with parafilm afterinoculum addition and monitored at 2, 7, 14, 21, 28 and 35 days. Platesthat showed contamination with bacteria or fungi were discarded.Nematode-induced infection galls were visible under low-powermagnification at 7 days, and adult females were visible at 25-30 days.

Scoring of Infection Assays

Gall number: The number of galls per plate was determined after 30-35days by counting under low-power magnification. Total number of galls,as well as the number of adult and gravid females, was recorded.Alternatively, total number of M. incognita at all stages was determinedby fuchsin staining of the roots [Eisenback (2000) Techniques formeasuring nematode development and egg production. in LaboratoryTechniques in Nematode Ecology. Wheeler et al., eds. Society ofNematologists: Hyattsville, Md. p. 1-4].

Brood size: Gravid females were excised from each separate assay plateand placed in microcentrifuge tubes. 1 mL of 10% bleach was added toeach tube and the tubes were agitated for 3 minutes. Freed eggs werecollected by microcentrifugation (1000×g, 2 minutes), rinsed three timeswith sterile water, and counted as described above. Brood size wasrecorded as eggs/female.

Brood viability: After counting, eggs from individual plates weretransferred in 500 μL water to wells of a 24-well plate and incubated atroom temperature in the dark for 7 days. The number of newly hatched J2larvae visible after this period was determined and recorded. Ability ofeggs or larvae to re-infect hairy roots was determined by inoculatingcontrol roots with eggs or J2's as described.

Scoring system based on root galling: A relatively higher throughputscoring system can be utilized when the number of plates becomesdifficult to score by the methods listed above. The following table isan example of a rating system based on visual estimation of root damagedcaused by Meloidogyne spp:

Damage Score Description 0 No galls 1 1-2 small galls 3 3-5 small galls5 >5 small galls, but no multiple galls 10 Several small galls and atleast one multiple gall 25 About 25% of the roots with multiple galls;many small galls 50 About 50% of the roots with multiple galls 75 About75% of the roots with multiple galls 90 Entire root system is galled andstunted

Soybean Cyst Nematode Pot Assay

This assay is used to evaluate the resistance of soybean plants toinfection by and reproduction of the soybean cyst nematode (Heteroderaglycines) on roots. Three or four inch diameter square pots were filledwith clean sand and watered thoroughly. Soybean seeds, or alternativelyany rooted plant parts, were planted one per pot in the center of thepot and watered well to remove air pockets. The pots were incubated inthe greenhouse or growth chamber at 20° C. to 30° C. until the plantsreached a suitable age for inoculation. Soybeans started from seed weretypically inoculated 2-3 weeks after planting, while transplants wereinoculated 1-3 days after planting. The test inoculum consisted of eggsfrom ripe H. glycines cysts collected from the soil and roots ofinfested soybean plants. A 250 micron mesh sieve was used to collect thecysts, which were then crushed in a Tenbroeck glass tissue homogenizerto release the eggs. The eggs were further purified by sieving andcentrifugation over 40% sucrose solution at 4000 RPM for 5 minutes.Inoculum for an experiment consisted of water containing 500 vermiformeggs per mL. Five mL of the egg suspension was pipetted over the surfaceof the sand containing the test plants and the eggs were lightly wateredin. The test plants were then returned to the greenhouse or growthchamber and incubated for 3-4 weeks to allow for root infection and cystformation. The roots were then harvested by gently removing the pot andsand and rinsing in water. The severity of nematode infection wasmeasured by counting the number of white nematode cysts adhering to theroot system. Alternatively, the sand and roots could be diluted in waterand passed over a 250 micron sieve to collect and concentrate the cystsfor storage or counting.

Use of tomato hairy roots for assay of cyst nematode infections: Theassay described above can also be used to determine the ability of cystnematode to infect tomato roots using the cyst nematode strain TN2.

EXAMPLE 19

TABLE 18 Sequence ID numbers for hydroxylase and epoxygenase genesConstruct cDNA Amino acid Ricinus communis SEQ ID NO: 1 SEQ ID NO: 13Lesquerella fendleri SEQ ID NO: 2 SEQ ID NO: 14 Lesquerella lindheimeriSEQ ID NO: 3 SEQ ID NO: 15 Lesquerella gracilis A SEQ ID NO: 4 SEQ IDNO: 16 Lesquerella gracilis B SEQ ID NO: 5 SEQ ID NO: 17 Crepis biennisSEQ ID NO: 6 SEQ ID NO: 18 fad2/R. communis SEQ ID NO: 7 SEQ ID NO: 19fad2/L. fendleri SEQ ID NO: 8 SEQ ID NO: 20 fad2/L. lindheimeri SEQ IDNO: 9 SEQ ID NO: 21 fad2/L. gracilis A SEQ ID NO: 10 SEQ ID NO: 22fad2/L. gracilis B SEQ ID NO: 11 SEQ ID NO: 23 fad2/C. biennis SEQ IDNO: 12 SEQ ID NO: 24 R. communis ΔKKGG SEQ ID NO: 25 SEQ ID NO: 34 L.gracilis B ΔT SEQ ID NO: 26 SEQ ID NO: 35 Stokesia laevis SEQ ID NO: 27SEQ ID NO: 36 R. communis optimization 2 SEQ ID NO: 28 SEQ ID NO: 37 S.laevis A optimization 2 SEQ ID NO: 29 SEQ ID NO: 38 R. communisoptimization 1 SEQ ID NO: 30 SEQ ID NO: 39 L. gracilis B optimization 1SEQ ID NO: 31 SEQ ID NO: 40 C. biennis optimization 1 SEQ ID NO: 32 SEQID NO: 41 S. laevis A optimization 1 SEQ ID NO: 33 SEQ ID NO: 42 HA R.communis optimization SEQ ID NO: 129 SEQ ID NO: 134 C. palaestinaoptimization SEQ ID NO: 130 SEQ ID NO: 135 S. laevis B optimization SEQID NO: 131 SEQ ID NO: 136 C. biennis optimization 2 SEQ ID NO: 132 SEQID NO: 137 L. gracilis B optimization 2 SEQ ID NO: 133 SEQ ID NO: 138Arabidopsis thaliana FAD2 5′-untranslated region (SEQ ID NO: 43 and 44)and Arabidopsis thaliana FAD2 3′-untranslated region (SEQ ID NO: 45).

EXAMPLE 20

This example describes the results of fatty acid analyses for tomatohairy roots and Arabidopsis thaliana seeds expressing variouscodon-optimized Ricinus communis constructs.

The fatty acid analysis of tomato hairy roots was carried out with thebasic derivatization method. Results of the analysis of tomato hairyroots expressing the SID 129 gene (the HA-tagged R. communissequence—SEQ ID NO: 129) are presented in Table 19. Results of theanalysis of tomato hairy roots expressing the SID 30 gene (of R.communis—SEQ ID NO: 30) or the SID 28 gene (of R. communis—SEQ ID NO:28) are presented in Table 20. Roots utilized in the analysis were grownunder light and temperature cycling conditions (12 hours at 23° C. inthe light alternating with 12 hours at 20° C. in the dark). A basicderivatization method was performed essentially as described by Cahoonet al. (Plant Physiol. 2002, 128: 615-624). Ground root tissue wasmethylated with 500 μL 1% sodium methoxide in methanol, extracted withhexane, and trimethylsilylated (100 μL BSTAFA-TMCS, Supelco, 90° C. for45 minutes). Samples were analyzed on an Agilent 6890 GC-5973 MassSelective Detector (GC/MS) and an Agilent DB-23 capillary column (0.25mm×30 m×0.25 um). The injector was held at 250° C., the oven temperaturewas 235° C., and a helium flow of 1.0 mL/min was maintained.

The fatty acid analysis of A. thaliana seeds was carried out with eitherthe basic or the acidic derivatization method. Results of the analysisof A. thaliana seeds expressing the SID 129 gene (the HA-tagged R.communis sequence—SEQ ID NO: 129) are presented in Table 21. Arabidopsisplants were grown in 3-inch pots under controlled environment in growthchambers. A temperature of 23° C. was maintained, with a 12 hour light:12 hour dark cycle. Plants were watered daily with tap water andfertilized once a week. The basic derivatization method was performedessentially as described by Cahoon et al. (Plant Physiol. 2002, 128:615-624). The acidic derivatization protocol is the same as the basicderivatization method, except that 500 μL 2.5% sulfuric acid in methanolis used in place of the sodium methoxide in methanol.

TABLE 19 Fatty acid analysis of tomato hairy roots Gene Line 18:1-OH18:2 18:1 16:0 18:0 18:3 SID 129 A .91 52.81 1.04 15.30 1.47 21.88 SID129 A 1.29 54.23 1.28 16.35 3.34 16.19 SID 129 B 0.92 54.00 4.62 14.052.62 18.28 SID 129 B 1.71 53.76 4.35 12.87 1.24 17.83 SID 129 C 1.2448.96 1.53 15.75 2.98 22.07 SID 129 C 2.5 54.69 2.2 15.11 2.51 17.60 SID129 D 3.03 51.41 1.74 14.90 4.46 15.96 SID 129 E 0.79 53.30 1.18 13.822.79 22.46 SID 129 F 0.93 57.49 2.3 14.22 2.42 18.51 EV G 0 58.01 1.1614.85 2.48 18.14 EV H 0 58.29 .60 15.81 2.35 18.03 SID 129: HA-tagged R.communis (SEQ ID NO: 129) basic derivatization method; EV: empty vector;18:1-OH - ricinoleic acid, 18:2 - linoleic acid; 18:1 - oleic acid;16:0 - palmitic acid; 18:0 - stearic acid; 18:3 - alpha linolenic acid.

TABLE 20 Fatty acid analysis of tomato hairy roots Gene Line 18:1-OH18:2 18:1 16:0 18:0 18:3 SID 30 A 2.76 50.95 5.10 16.17 3.06 14.39 SID30 B 1.34 54.78 4.53 14.26 1.25 14.99 SID 30 C 3.21 51.75 3.89 14.032.07 16.41 SID 30 C 2.215 50.24 3.45 15.51 2.86 15.81 SID 30 D 3.0451.71 8.89 14.26 2.33 11.71 SID 28 A 3.23 48.70 1.70 12.92 3.40 17.22SID 28 A 3.65 51.59 2.79 11.23 1.48 21.54 SID 28 B 2.98 51.38 2.97 12.892.97 19.48 SID 28 B 1.56 51.37 1.96 14.33 2.78 19.95 SID 28 C 2.48 54.404.36 14.20 1.19 17.22 SID 28 D 4.73 54.69 2.22 10.05 2.83 18.04 SID 28 D3.32 55.17 2.49 12.89 3.29 16.07 SID 28 E 2.847 52.46 2.25 12.05 2.6119.06 SID 28 F 1.96 55.91 2.55 14.50 2.88 15.81 EV G 0 56.31 0.964 15.941.61 19.48 EV G 0 56.3 1.6 15.96 1.61 19.45 SID 30: R. communis (SEQ IDNO: 30) basic derivatization method; SID 28: R. communis (SEQ ID NO: 28)basic derivatization method

TABLE 21 Fatty acid analysis of A. thaliana seeds 18:1- 18:2- 20:1- Gene18:1 18:2 16:0 18:0 18:3 20:0 OH OH OH SID 129 21.19 20.04 7.99 4.212.01 4.39 3.78 1.15 1.02 A SID 129 21.16 21.97 9.15 3.79 13.81 2.472.29 1.42 0.81 A EV A 20.95 27.25 6.41 3.53 14.56 1.89 0 0 0 SID 12921.45 20.82 9.68 3.76 13.2 2.64 2.64 1.7 0.84 B SID 129 20.51 22.97 7.623.01 14.05 1.67 1.75 1.65 0.66 B SID 129 20.43 23.07 7.78 2.99 13.971.66 1.6 1.64 0.62 B EV B 22.67 28.43 6.13 2.61 14.56 1.9 0 0 0 SID 129A or B: HA-tagged R. communis (SEQ ID NO: 129) acidic or basicderivatization methods, respectively; EV A or B: empty vector acidic orbasic derivatization methods, respectively; 18:1 - oleic acid, 18:2 -linoleic acid, 16:0 - palmitic acid, 18:0 - stearic acid, 18:3 - alphalinolenic acid; 20:0 - arachidic acid, 18:1-OH - ricinoleic acid,18:2-OH - densipolic acid, 20:0-OH - lesquerolic acid.

Tables 19 and 20 show that codon optimization of castor genes allows foran accumulation of ricinoleic acid (18:1-OH) in vegetative tissues ofplants expressing such genes, as compared to no accumulation in plantstransformed with an empty vector. Table 21 shows that the ricinoleicacid accumulation is detected in A. thaliana seeds, even though the CaMV35S promoter is not a seed specific promoter. Taken together, theresults of these and the experiments described above suggest that anincreased accumulation of novel fatty acids in transgenic plants isuseful for both nematode control as well as for non-pesticidalindustrial uses (e.g., in oil seed engineering).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A transgenic plant containing at least one DNA construct, saidconstruct comprising: a) a nucleic acid encoding a polypeptide effectivefor catalysing the conversion of a substrate to a C16, C18, or C20monounsaturated fatty acid product, wherein said polypeptide has atleast 95% sequence identity to the amino acid sequence shown in SEQ IDNO: 136, wherein said fatty acid product has the following structure:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na+, phosphatidylcholine, or phosphatidylethanolamine, andwherein R3 is C2, C4, or C6 alkyl; and (b) a regulatory element operablylinked to said nucleic acid encoding said polypeptide, wherein saidregulatory element confers expression in a vegetative tissue of saidplant, and wherein said transgenic plant has anthelmintic activity. 2.The plant according to claim 1, wherein the double bond between the 9thand 10th carbons is cis.
 3. The plant according to claim 1, wherein thedouble bond between the 9th and 10th carbons is trans.
 4. The plantaccording to claim 1, wherein said regulatory element is a 5′-regulatoryelement.
 5. The plant of claim 4, wherein said 5′-regulatory elementconfers expression in root tissue.
 6. The plant of claim 5, said planthaving a significantly increased amount of a epoxy-fatty acid in rootsof said plant relative to a corresponding plant that lacks said DNAconstruct.
 7. The plant of claim 6, wherein said epoxy-fatty acid isvernolic acid.
 8. The plant of claim 7, wherein said vernolic acidconstitutes from about 0.1% to about 25% of the total fatty acid contentof said roots.
 9. The plant of claim 4, wherein said 5′-regulatoryelement is selected from the group consisting of a potato ribosomalprotein S27a Ubi3 promoter, a RB7 promoter, an alfalfa histone H3.2promoter, an IRT2 promoter, an Arabidopsis FAD2 5′-UTR, an ArabidopsisFAD3 5′-UTR, a Ubi3 5′-UTR, an alfalfa histone H3.2 5′-UTR, and aCaMV35S 5′-UTR.
 10. The plant of claim 1, wherein said regulatoryelement comprises a first 5′-regulatory element operably linked to asecond 5′-regulatory element, wherein said first 5′-regulatory elementis an Ubi3 promoter and said second 5′-regulatory element is selectedfrom the group consisting of an Arabidopsis FAD2 5′-UTR, an ArabidopsisFAD3 5′-UTR, a potato ribosomal protein S27a 5′-UTR, a Ubi3 5′-UTR, anda CaMV35S 5′-UTR.
 11. The plant of claim 4, wherein said DNA constructfurther comprises a 3′-regulatory element.
 12. The plant of claim 11,wherein said 3′-regulatory element comprises a Ubi3 terminator or an E9pea terminator.
 13. The plant of claim 11, wherein said 5′-regulatoryelement is selected from the group consisting of an Arabidopsis FAD25′-UTR and an Arabidopsis FAD3 5′-UTR and said 3′-regulatory element isselected from the group consisting of an Arabidopsis FAD2 3′-UTR and anArabidopsis FAD3 3′-UTR.
 14. The plant of claim 13, wherein said5′-regulatory element comprises SEQ ID NOS: 43 or 44 and said3′-regulatory element comprises SEQ ID NO:
 45. 15. The plant of claim 1,wherein said at least one DNA construct further comprises at least oneregulatory element that confers expression in vegetative tissues of aplant operably linked to a nucleic acid that encodes a PDAT or DAGATpolypeptide.
 16. The plant of claim 1, said plant further comprising asecond DNA construct, said second DNA construct comprising at least oneregulatory element that confers expression in vegetative tissues of aplant operably linked to a nucleic acid that encodes a PDAT or DAGATpolypeptide.
 17. The plant of claim 1, wherein R3 is C2 alkyl or C4alkyl.
 18. The plant of claim 1, where said plant is selected from thegroup consisting of tobacco, tomato, soybean, corn, cotton, rice, wheat,banana, carrot, potato, strawberry and turf grass.
 19. A method ofmaking a transgenic plant having anthelmintic activity, said methodcomprising introducing a construct into a plant, wherein said constructcomprises: a) a nucleic acid encoding a polypeptide effective forcatalysing the conversion of a substrate to a C16, C18, or C20monounsaturated fatty acid product, wherein said polypeptide has theamino acid sequence shown in SEQ ID NO: 136, wherein said fatty acidproduct has the following structure:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na⁺, phosphatidylcholine, or phosphatidylethanolamine, andwherein R₃ is C2, C4, or C6 alkyl; and (b) a regulatory element operablylinked to said nucleic acid encoding said polypeptide, wherein saidregulatory element confers expression in a vegetative tissue of saidplant.
 20. The method of claim 19, wherein said regulatory element ofsaid construct is a 5′-regulatory element.
 21. The method of claim 19,wherein said 5′-regulatory element comprises a potato ribosomal proteinS27a Ubi3 promoter, a RB7 promoter, an alfalfa histone H3.2 promoter, anIRT2 promoter, an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, aUbi3 5′-UTR, an alfalfa histone H3.2 5′-UTR, and a CaMV35S 5′-UTR. 22.The method of claim 19, wherein said regulatory element comprises afirst 5′-regulatory element operably linked to a second 5′-regulatoryelement, wherein said first 5′-regulatory element is an Ubi3 promoterand said second 5′-regulatory element is selected from the groupconsisting of an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, apotato ribosomal protein S27a 5′-UTR, a Ubi3 5′-UTR, and a CaMV35S5′-UTR.
 23. The method of claim 20, wherein said DNA construct furthercomprises a 3′-regulatory element.
 24. The method of claim 23, whereinsaid 5′-regulatory element comprises SEQ ID NO: 43 or SEQ ID NO: 44 andsaid 3′-UTR comprises SEQ ID NO:
 45. 25. An isolated nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO:131.
 26. Arecombinant nucleic acid construct comprising at least one regulatoryelement that confers expression in a vegetative tissue of a plant, saidregulatory element operably linked to a nucleic acid having thenucleotide sequence set forth in SEQ ID NO:131.
 27. The nucleic acidconstruct of claim 26, wherein said at least one regulatory elementcomprises a 5′-regulatory element having the nucleotide sequence setforth in SEQ ID NO: 43 or SEQ ID NO:
 44. 28. The nucleic acid constructof claim 27, wherein said construct further comprises a 3′-regulatoryelement having the nucleotide sequence set forth in SEQ ID NO:
 45. 29. Atransgenic plant harboring a DNA construct comprising a nucleic acidencoding a fatty acid epoxygenase polypeptide operably linked to aregulatory element conferring expression of said polypeptide in avegetative tissue of said plant, wherein said polypeptide has the aminoacid sequence shown in SEQ ID NO:
 136. 30. The plant of claim 29, saidplant having a significantly increased amount of a epoxy-fatty acid inroots of said plant relative to a corresponding plant that lacks saidDNA construct.
 31. The plant of claim 30, wherein said epoxy-fatty acidis vernolic acid.
 32. The plant of claim 31, wherein said vernolic acidconstitutes from about 0.1% to about 25% of the total fatty acid contentof said roots.
 33. A transgenic plant containing at least one DNAconstruct, said construct comprising: a) a nucleic acid encoding apolypeptide effective for catalysing the conversion of a substrate to aC16, C18, or C20 monounsaturated fatty acid product, wherein saidpolypeptide has at least 95% sequence identity to the amino acidsequence shown in SEQ ID NO: 136, wherein said fatty acid product hasthe following structure:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na⁺, phosphatidylcholine, or phosphatidylethanolamine, andwherein R3 is C2, C4, or C6 alkyl; and (b) a regulatory element operablylinked to said nucleic acid encoding said polypeptide, wherein saidregulatory element confers expression in at least one tissue of seeds ofsaid plant, and wherein said transgenic plant has anthelmintic activity.34. The plant according to claim 33, wherein said regulatory element isa 5′-regulatory element.
 35. The plant of claim 34, said plant having asignificantly increased amount of an epoxy-fatty acid in at least onetissue of seeds of said plant relative to a corresponding plant thatlacks said DNA construct.
 36. The plant of claim 35, wherein saidepoxy-fatty acid is vernolic acid.
 37. A method of making a transgenicplant having anthelmintic activity, said method comprising introducing aconstruct into a plant, wherein said construct comprises: a) a nucleicacid encoding a polypeptide effective for catalysing the conversion of asubstrate to a C16, C18, or C20 monounsaturated fatty acid product,wherein said polypeptide has the amino acid sequence shown in SEQ ID NO:136, wherein said fatty acid product has the following structure:

wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride,ACP, methyl, Na+, phosphatidylcholine, or phosphatidylethanolamine, andwherein R₃ is C2, C4, or C6 alkyl; and (b) a regulatory element operablylinked to said nucleic acid encoding said polypeptide, wherein saidregulatory element confers expression in a vegetative tissue of saidplant.